U.S. patent application number 10/772437 was filed with the patent office on 2004-10-14 for asthma associated factors as targets for treating atopic allergies including asthma and related disorders.
This patent application is currently assigned to Magainin Pharmaceuticals Inc.. Invention is credited to Dong, Qu, Holroyd, Kenneth J., Levitt, Roy C., Louahed, Jamila, Maloy, W. Lee, McLane, Mike, Nicolaides, Nicholas C., Zhou, Yuhong.
Application Number | 20040204578 10/772437 |
Document ID | / |
Family ID | 22134353 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040204578 |
Kind Code |
A1 |
Holroyd, Kenneth J. ; et
al. |
October 14, 2004 |
Asthma associated factors as targets for treating atopic allergies
including asthma and related disorders
Abstract
A new gene in the calcium-activated chloride channel family has
been discovered that is induced by IL-9, thereby providing a
therapeutic target in IL-9 mediated development of atopic allergy,
asthma-related disorders and cystic fibrosis. A method for the
identification and use of small molecule inhibitors of this gene
and its products to treat these disorders has also been discovered.
The invention also includes a method for diagnosing susceptibility
to, and assessing treatment of atopic allergy, asthma-related
disorders by measuring the level of gene expression in biologic
samples using antibody specific for this protein.
Inventors: |
Holroyd, Kenneth J.;
(Plymouth Meeting, PA) ; Levitt, Roy C.; (Plymouth
Meeting, PA) ; Maloy, W. Lee; (Plymouth Meeting,
PA) ; Louahed, Jamila; (Plymouth Meeting, PA)
; McLane, Mike; (Plymouth Meeting, PA) ;
Nicolaides, Nicholas C.; (Boothwyn, PA) ; Zhou,
Yuhong; (Plymouth Meeting, PA) ; Dong, Qu;
(Dresher, PA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Magainin Pharmaceuticals
Inc.
|
Family ID: |
22134353 |
Appl. No.: |
10/772437 |
Filed: |
February 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10772437 |
Feb 6, 2004 |
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10270595 |
Oct 16, 2002 |
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6716603 |
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10270595 |
Oct 16, 2002 |
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09623624 |
Feb 13, 2001 |
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6576434 |
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09623624 |
Feb 13, 2001 |
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PCT/US99/04703 |
Mar 3, 1999 |
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60076815 |
Mar 3, 1998 |
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Current U.S.
Class: |
536/23.2 |
Current CPC
Class: |
G01N 2500/00 20130101;
C07K 14/705 20130101; Y10S 977/927 20130101; A61K 38/00 20130101;
G01N 33/6872 20130101; A61P 11/00 20180101; A61P 37/08 20180101;
A61P 43/00 20180101; A61P 1/00 20180101; A61P 11/06 20180101; Y10S
977/914 20130101 |
Class at
Publication: |
536/023.2 ;
514/044 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
1. A purified and isolated nucleic acid molecule selected from the
group consisting of a nucleic acid molecule having a nucleotide
sequence encoding human ICACC-1 (SEQ ID NO: 6), a nucleic acid
molecule which hybridizes to a nucleic acid molecule having SEQ ID
NO: 1 and a nucleic acid molecule comprising functionally effective
fragments thereof.
2. to 36. (cancelled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Serial No. 60/076,815 which was filed Mar. 3,
1998 and which is herein incorporated by reference in its entirety.
This invention is also related to the subject matter of U.S. patent
application Ser. Nos. 08/697,419; 08/697,360; 08/697,473;
08/697,472; 08/697,471; 08/702,105; 08/702,110; 08/702,168; and
08/697,440, all of which were filed on Aug. 23, 1996 and are
incorporated herein by reference. This application is also related
to U.S. patent application Ser. No. 08/980,872 which was filed Dec.
1, 1997 and which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to modulating activities associated
with the IL-9 pathway for the treatment of atopic allergies and
related disorders such as asthma.
BACKGROUND OF THE INVENTION
[0003] Inflammation is a complex process in which the body's
defense system combats foreign entities. While the battle against
foreign entities may be necessary for the body's survival, some
defense systems respond to foreign entities, even innocuous ones,
as dangerous and thereby damage surrounding tissue in the ensuing
battle.
[0004] Atopic allergy is an ecogenetic disorder, where genetic
background dictates the response to environmental stimuli. The
disorder is generally characterized by an increased ability of
lymphocytes to produce IgE antibodies in response to ubiquitous
antigens. Activation of the immune system by these antigens leads
to allergic inflammation and may occur after ingestion, penetration
through the skin or after inhalation. When this immune activation
occurs and is accompanied by pulmonary inflammation and bronchial
hyperresponsiveness, this disorder is broadly characterized as
asthma. Certain cells are important in this inflammatory reaction
and they include T cells and antigen-presenting cells, B cells that
produce IgE, basophils that bind IgE and eosinophils. These
inflammatory cells accumulate at the site of allergic inflammation
and the toxic products they release contribute to tissue
destruction related to these disorders.
[0005] While asthma is generally defined as an inflammatory
disorder of the airways, clinical symptoms arise from intermittent
air flow obstruction. It is a chronic, disabling disorder that
appears to be increasing in prevalence and severity (Gergen et al.,
1992). It is estimated that 30-40% of the population suffer with
atopic allergy and 15% of children and 5% of adults in the
population suffer from asthma (Gergen et al., 1992). Thus, an
enormous burden is placed on health-care resources.
[0006] Interestingly, while most individuals experience similar
environmental exposures, only certain individuals develop atopic
allergy and asthma. This hypersensitivity to environmental
allergens known as "atopy" is often indicated by elevated serum IgE
levels or abnormally intense skin test response to allergens in
atopic individuals as compared to nonatopics (Marsh et al., 1982).
Strong evidence for a close relationship between atopic allergy and
asthma is derived from the fact that most asthmatics have clinical
and serologic evidence of atopy (Clifford et al., 1987; Gergen,
1991; Burrows et al., 1992; Johannson et al., 1972; Sears et al.,
1991; Halonen et al., 1992). In particular, younger asthmatics have
a high incidence of atopy (Marsh et al., 1982). In addition,
immunologic factors associated with an increase in total serum IgE
levels are very closely related to impaired pulmonary function
(Burrows et al., 1989).
[0007] Both the diagnosis and treatment of these disorders are
problematic (Gergen et al., 1992). The assessment of inflamed lung
tissue is often difficult and frequently the source of the
inflammation cannot be determined. It is now generally accepted
that failure to control pulmonary inflammation leads to significant
loss of lung function over time.
[0008] Current treatments suffer their own set of disadvantages.
The main therapeutic agents, .beta. agonists, reduce the symptoms
thereby transiently improving pulmonary function, but do not affect
the underlying inflammation so that lung tissue remains in
jeopardy. In addition, constant use of .beta. agonists results in
desensitization which reduces their efficacy and safety (Molinoff
et al., 1995). The agents that can diminish the underlying
inflammation, such as anti-inflammatory steroids, have their own
list of disadvantages that range from immunosuppression to bone
loss (Molinoffet al., 1995).
[0009] Because of the problems associated with conventional
therapies, alternative treatment strategies have been evaluated.
Glycophorin A (Chu et al., 1992), cyclosporin (Alexander et al.,
1992; Morely, 1992) and a nonapeptide fragment of interleukin 2
(IL-2) (Zavyalov et al., 1992) all inhibit potentially critical
immune functions associated with homeostasis. What is needed in the
art is a treatment for asthma that addresses the underlying
pathogenesis. Moreover, these therapies must address the edisodic
nature of the disorder and the close association with allergy and
intervene at a point downstream from critical immune functions.
[0010] In the related patent applications mentioned above,
applicants have demonstrated that interleukin 9 (IL-9), its
receptor and activities effected by IL-9 are the appropriate
targets for therapeutic intervention in atopic allergy, asthma and
related disorders.
[0011] Mediator release from mast cells by allergen has long been
considered a critical initiating event in allergy. IL-9 was
originally identified as a mast cell growth factor and it has been
demonstrated that IL-9 up-regulates the expression of mast cell
proteases including MCP-1, MCP-2, MCP-4 (Eklund et al., 1993) and
granzyme B (Louahed et al., 1995). Thus, IL-9 appears to serve a
role in the proliferation and differentiation of mast cells.
Moreover, IL-9 up-regulates the expression of the alpha chain of
the high affinity IgE receptor (Dugas et al., 1993). Elevated IgE
levels are considered to be a hallmark of atopic allergy and a risk
factor for asthma. Furthermore, both in vitro and in vivo studies
have shown IL-9 to potentiate the release of IgE from primed B
cells (Petit-Frere et al., 1993).
[0012] There is substantial support for the role of IL-9 gene in
asthma. First, linkage homology between humans and mice suggests
that the same gene is responsible for producing biologic
variability in response to antigen in both species. Second,
differences in expression of the murine IL-9 candidate gene are
associated with biologic variability in bronchial responsiveness.
In particular, reduced expression of IL-9 is associated with a
lower baseline bronchial response in B6 mice. Third, recent
evidence for linkage disequilibrium in data from humans suggests
IL-9 may be associated with atopy and bronchial hyperresponsiveness
consistent with a role for this gene in both species (Doull et al.,
1992). Moreover, a genetic alteration in the human gene appears to
be associated with loss of cytokine function and lower IgE levels.
Fourth, the pleiotropic functions of this cytokine and its receptor
in the allergic immune response strongly support a role for the
IL-9 pathway in the complex pathogenesis of asthma. Fifth, in
humans, biologic variability in the IL-9 receptor also appears to
be associated with atopic allergy and asthma. Finally, despite the
inherited loss of IL-9 receptor function, these individuals appear
to be otherwise healthy. Thus, nature has demonstrated in atopic
individuals that the therapeutic down-regulation of IL-9 and IL-9
receptor genes or genes activated by IL-9 and its receptor is
likely to be safe.
[0013] While the role of the IL-9 gene, its receptor and their
functions in atopic allergy, asthma and related disorders has been
elucidated, a specific need in the art exists for elucidation of
the role of genes which are regulated by IL-9 in the etiology of
these disorders. Furthermore, most significantly, based on this
knowledge, there is a need for the identification of agents that
are capable of regulating the activity of these genes or their gene
products for treating these disorders.
[0014] Cystic fibrosis is yet another disease which effects the
lung and is associated with thick secretions resulting in airway
obstruction and subsequent colonization and infection by inhaled
pathogenic microorganisms (Eng et al., 1996). Cystic fibrosis
airway epithelia exhibit a spectrum of ion transport properties
that differ from normal, including not only defective cAMP-mediated
chloride secretion, but also increased sodium absorption and
increased calcium-mediated chloride secretion (Johnson et al.,
1995). The increase in calcium-mediated chloride secretion is
presumably an attempt to compensate for the overall decrease in
chloride secretion due to the defect in cAMP-mediated chloride
secretion. It does not adequately compensate for this defect,
however, because normal chloride gradients are not maintained.
Thus, potential therapeutic remedies for cystic fibrosis rely on
mechanisms which increase chloride secretion in airway epithelial
cells to compensate for defective cAMP-mediated chloride secretion.
Such mechanisms are capable of restoring the cellular chloride
gradient thereby alleviating sodium hyperabsorption associated with
decreased chloride secretion. A specific need in the art therefore
exists for identification of agents capable of enhancing
calcium-dependent chloride secretion in cystic fibrosis airway
epithelial cells.
SUMMARY OF THE INVENTION
[0015] The present invention includes new genes from the calcium
activated chloride channel gene family designated ICACC (IL-9
Induced Calcium Activated Chloride Channel), particularly the mouse
(SEQ ID NO:1) and human (SEQ ID NO:3 and SEQ ID NO:5) ICACC genes.
The ICACC-1 genes are selectively up-regulated by IL-9 and
therefore part of the IL-9 signaling pathway. The present invention
also includes the protein products of the ICACC genes,
particularly, the mouse (SEQ ID NO:2) and human (SEQ ID NO:4 and
NO:6) ICACC genes.
[0016] The inventors have satisfied the need for diagnosis and
treatment of atopic allergy, asthma and related disorders by
demonstrating the role of ICACC-1 in the pathogenesis of these
disorders. Therapies for these disorders are derived from the
down-regulation of ICACC-1 as a member of the IL-9 pathway.
[0017] The identification of ICACC-1 has led to the discovery of
compounds that are capable of down-regulating its activity.
Activity is defined here as any alteration in either chloride
channel function or expression of ICACC-1. Molecules that
down-regulate ICACC-1 are therefore part of the invention.
Down-regulation is defined here as a decrease in activation,
function or synthesis of ICACC-1, its ligands or activators. It is
further defined to include an increase in the degradation of
ICACC-1 gene, its protein product, ligands or activators.
Down-regulation is therefore achieved in a number of ways. For
example, administration of molecules that can destabilize the
binding of ICACC-1 with its ligands. Such molecules encompass
polypeptide products, including those encoded by the DNA sequences
of the ICACC-1 gene or DNA sequences containing various mutations.
These mutations may be point mutations, insertions, deletions or
spliced variants of the ICACC-1 gene. This invention also includes
truncated polypeptides encoded by the DNA molecules described
above. These polypeptides being capable of interfering with
interaction of ICACC-1 with its ligand and other proteins.
[0018] A further embodiment of this invention includes the
down-regulation of ICACC-1 function by altering expression of the
ICACC-1 gene, the use of antisense gene therapy being an example.
Down-regulation of ICACC-1 expression is accomplished by
administering an effective amount of antisense oligonucleotides.
These antisense molecules can be fashioned from the DNA sequence of
the ICACC-1 gene or sequences containing various mutations,
deletions, insertions or spliced variants. Another embodiment of
this invention relates to the use of isolated RNA or DNA sequences
derived from the ICACC-1 gene. These sequences containing various
mutations such as point mutations, insertions, deletions or spliced
variant mutations of ICACC-1 gene and can be useful in gene
therapy.
[0019] Molecules that increase the degradation of the ICACC-1
protein may also be used to down-regulate its functions and are
within the scope of the invention. Phosphorylation of ICACC-1 may
alter protein stability, therefore kinase inhibitors may be used to
down-regulate its function. Down-regulation of ICACC-1 may also be
accomplished by the use of polyclonal or monoclonal antibodies or
fragments thereof directed against the ICACC-1 protein. Such
molecules are within the claimed invention. This invention further
includes small molecules with the three-dimensional structure
necessary to bind with sufficient affinity to block ICACC-1
interactions with its ligands or block function of the chloride
channel. ICACC-1 blockade resulting in deregulation of calcium and
chloride flux and other processes of proinflammatory cells where it
is expressed make these small molecules useful as therapeutic
agents in treating inflammation associated with atopic allergy,
asthma and related disorders. In a further embodiment, aminosterol
compounds are assessed for their ability to block ICACC-1 induction
by IL-9 or antigen as a means of determining their usefulness in
treating atopic allergies and related disorders.
[0020] The agents discussed above represent various effective
therapeutic compounds in treating atopic allergies, asthma and
other related disorders. Applicants have thus provided antagonists
and methods of identifying antagonists that are capable of
down-regulating ICACC-1. Applicants also provide methods for
down-regulating the activity of ICACC-1 by administering truncated
protein products, chloride channel blockers, aminosterols and the
like.
[0021] Applicants also provide a method for the diagnosis of
susceptibility to atopic allergy, asthma and related disorders by
describing a method for assaying the induction of ICACC-1, its
functions or downstream activities. In a further embodiment,
Applicants provide methods to monitor the effects of ICACC-1
down-regulation as a means to follow the treatment of atopic
allergy and asthma. Applicants also provide a method for diagnosing
autoimmune type diseases such as inflammatory bowel disease (IBD)
where suppression of TH2-associated responses (such as the biologic
responses associated with IL-9) are a common molecular feature. The
constitutive expression of ICACC-1 in the small intestine and colon
suggest that this is a useful marker for monitoring treatment of
TH1 associated disease states such as IBD, where down regulated
expression of ICACC-1 will be associated with the disease.
[0022] In a further embodiment, Applicants identify a disease
state, which can be treated through the up-regulation of ICACC-1.
Applicants provide a method for treating the defect in
cAMP-mediated chloride secretion in cystic fibrosis airway
epithelia by further increasing calcium-dependent chloride
secretion through up-regulation of ICACC-1. This up-regulation of
ICACC-1 resulting in increased chloride secretion and thus
restoration of the cellular chloride gradient resulting in normal
airway epithelial cell function. Applicants provide a method for
treating inflammatory bowel disease (IBD) with local delivery of
ICACC-1 via gene therapy or up regulation of ICACC-1 to enhance
TH2-associated responses for suppressing the TH1-associated IBD
autoimmune disease.
[0023] The accompanying figures, which are incorporated in and
constitute apart of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principle of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 shows a schematic diagram of the suppressive PCR cDNA
subtraction technique.
[0025] FIG. 2 shows the nucleotide (SEQ ID NO:1) and amino acid
(SEQ ID NO:2) sequence of the murine ICACC-1 cDNA.
[0026] FIG. 3 shows an alignment of the murine ICACC-1 protein with
a bovine calcium activated chloride channel.
[0027] FIG. 4A shows the nucleotide (SEQ ID NO:3) and amino acid
(SEQ ID NO:4) sequence of the human ICACC-2 cDNA.
[0028] FIG. 4B shows the nucleotide (SEQ ID NO:5) and amino acid
(SEQ ID NO:6) sequence of the human ICACC-1 cDNA.
[0029] FIG. 5 shows an alignment of the murine ICACC-1 protein with
the human ICACC-1 and ICACC-2 protein.
[0030] FIG. 6 shows ICACC-1 expression in the lung of normal mice
(FVB) compared to transgenic mice overexpressing the IL-9 gene
(Tg5).
[0031] FIG. 7 shows the expression of ICACC-1 in the lungs of DBA
and C57B6 mice.
[0032] FIG. 8 shows the expression of ICACC-1 in the lung of the
C57B6 mouse with and without intratracheal administration of
IL-9.
[0033] FIG. 9 shows the expression of ICACC-1 in tissues from
normal (Balb/C) and IL-9 overexpressing (Tg5) mice.
[0034] FIG. 10 shows Aspergillus fumagatus-antigen induced BHR and
eosinophilia in Balb/C mice.
[0035] FIG. 11 shows the tissue distribution of ICACC-1 in naive
and antigen exposed Balb/C mice.
[0036] FIG. 12 shows the suppression of BHR and lung eosinophilia
by anti-IL9 in mice exposed to Aspergillus fumagatus.
[0037] FIG. 13 shows suppression of ICACC-1 in antigen exposed
animals treated with anti-IL9.
[0038] FIG. 14 shows ICACC-1 induction by IL-9 in human primary
lung epithelial cells (NHBE).
[0039] FIG. 15 shows ICACC-1 induction by IL-9 in human primary
lung cultures.
[0040] FIG. 16 shows antisera generated against ICACC-1 peptides is
able to recognize native ICACC-1
[0041] FIG. 17 shows IL-9 induces eotaxin production from
epithelial cells in primary lung cultures
[0042] FIG. 18 shows suppression of IL-9 induced eotaxin by
chloride channel blockers
DETAILED DESCRIPTION OF THE INVENTION
[0043] The inventors have resolved a crucial need in the art by
elucidating critical genes in the IL-9 pathway and compositions
affecting that pathway which may be used in the diagnosis,
prevention or treatment of atopic allergy including asthma and
related disorders. Asthma encompasses inflammatory disorders of the
airways with reversible airflow obstruction. Atopic allergy refers
to atopy and related disorders including asthma, bronchial
hyperresponsiveness, rhinitis, urticaria, allergic inflammatory
disorders of the bowel and various forms of eczema. Atopy is a
hypersensitivity to environmental allergens expressed as the
elevation of serum total IgE or abnormal skin test responses to
allergens as compared to controls. Bronchial hyperresponsiveness is
a heightened broncho constrictor response to a variety of
stimuli.
[0044] A. ICACC Proteins
[0045] The present invention provides isolated ICACC protein,
allelic variants of the protein, and conservative amino acid
substitutions of the protein. As used herein, the ICACC protein or
polypeptide includes a protein that has the murine amino acid
sequence of SEQ ID NO: 2 or the human amino acid sequence depicted
in SEQ ED No.4 or SEQ ID No.6. The invention includes naturally
occurring allelic variants and proteins that have a slightly
different amino acid sequence than that specifically recited above.
Allelic variants, though possessing a slightly different amino acid
sequence than those recited above, will still have the same or
similar biological functions associated with the ICACC protein.
[0046] As used herein, the family of proteins related to the ICACC
protein refer to proteins that have been isolated from organisms in
addition to humans or mice. The methods used to identify and
isolate other members of the family of proteins related to the
human and/or murine ICACC proteins are described below.
[0047] The proteins of the present invention are preferably in
isolated form. As used herein, a protein is said to be isolated
when physical, mechanical or chemical methods are employed to
remove the protein from cellular constituents that are normally
associated with the protein. A partially isolated protein, as used
herein, includes ICACC proteins isolated in membrane fragments,
including cellular membrane fragments containing a recombinantly
expressed ICACC protein. A skilled artisan can readily employ
standard purification methods to obtain an isolated protein.
[0048] The proteins of the present invention further include
conservative variants of the proteins herein described. As used
herein, a conservative variant refers to alterations in the amino
acid sequence that do not adversely affect the biological functions
of the protein. A substitution, insertion or deletion is said to
adversely affect the protein when the altered sequence prevents or
disrupts a biological function associated with the protein. For
example, the overall charge, structure or hydrophobic/hydrophilic
properties of the protein may be altered without adversely
affecting a biological activity. Accordingly, the amino acid
sequence can be altered, for example to render the peptide more
hydrophobic or hydrophilic, without adversely affecting the
biological activities of the protein.
[0049] Ordinarily, the allelic variants, the conservative
substitution variants, and the members of the protein family, will
have an amino acid sequence having at least about 55%, at least
about 75% amino acid sequence identity with the murine sequence set
forth in SEQ ID No.2 or the human sequences of SEQ ID NO: 4 or SEQ
ID No.6, more preferably at least 80%, even more preferably at
least 90%, and most preferably at least 95%. Identity or homology
with respect to such sequences is defined herein as the percentage
of amino acid residues in the candidate sequence that are identical
with the known peptides, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology, and not considering any conservative substitutions as
part of the sequence identity. N-terminal, C-terminal or internal
extensions, deletions, or insertions into the peptide sequence
shall not be construed as affecting homology.
[0050] Thus, the proteins of the present invention include
molecules having the amino acid sequence disclosed in SEQ ID Nos.
2, 4 or 6; fragments thereof having a consecutive sequence of at
least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid
residues of the ICACC protein; amino acid sequence variants of such
sequence wherein an amino acid residue has been inserted N- or
C-terminal to, or within, the disclosed sequence; and amino acid
sequence variants of the disclosed sequence, or their fragments as
defined above, that have been substituted by another residue.
Contemplated variants further include those containing
predetermined mutations by, e.g., homologous recombination,
site-directed or PCR mutagenesis, and the corresponding proteins of
other animal species, including but not limited to rabbit, rat,
porcine, bovine, ovine, equine and non-human primate species, and
the alleles or other naturally occurring variants of the family of
proteins; and derivatives wherein the protein has been covalently
modified by substitution, chemical, enzymatic, or other appropriate
means with a moiety other than a naturally occurring amino acid
(for example a detectable moiety such as an enzyme or
radioisotope).
[0051] As described below, members of the family of proteins can be
used: 1) to identify agents which modulate at least one activity of
the protein; 2) in methods of identifying binding partners for the
protein; and 3) as an antigen to raise polyclonal or monoclonal
antibodies.
[0052] B. Nucleic Acid Molecules
[0053] The murine ICACC-1 gene was identified by subtractive cDNA
cloning experiments that were performed in order to identify genes
specifically induced by IL-9. A schematic diagram of the
subtractive cDNA cloning method is provided in FIG. 1. RNA derived
from lungs of transgenic mice overexpressing the murine IL-9
transgene (Tg5) was used to isolate genes expressed in response to
IL-9 as opposed to those which are not expressed in the parental
strain (FVB). FIG. 6 shows a Northern blot with RNA from a lung of
a Tg5 mouse (right lane) and a FVB mouse (left lane) demonstrating
these findings. Expression of ICACC-1 was also observed in the lung
of the DBA murine strain which has been shown to express elevated
baseline IL-9 levels in their lungs FIG. 7). ICACC-1 expression was
not observed in the lungs of the C57B6 strain where IL-9 expression
is below the limits of detection (FIG. 7) (Nicolaides et al.,
1997). The direct effect of IL-9 on inducing ICACC-1 expression was
demonstrated when IL-9 was instilled into the trachea of the C57B6
mouse. The results of this experiment demonstrated that ICACC-1 was
expressed in the lungs of the IL-9 instilled mice but not in naive
or vehicle treated mice (FIG. 8), indicating that this gene is
induced by IL-9. The results also show that ICACC-1 gene expression
is induced in the lung of antigen exposed mice which exhibit
asthmatic-like features (BHR, lung eosinophilia) (FIGS. 10 and 12).
The antigen induced BHR and lung eosinophilia can be suppressed in
mice by treatment with anti-IL9 (FIG. 12), which also results in
down regulation of ICACC-1 (FIG. 13).
[0054] The murine ICACC-1 gene displayed significant homology
(.about.50%) with a member of the bovine calcium activated chloride
channel family (FIG. 3) (Cunningham et al., 1995). The full length
cDNA was cloned from a murine cDNA library (FIG. 2). Several EST
were identified which displayed partial homology to the murine
ICACC-1. These EST were obtained from the IMAGE consortium
(Lawrence Livermore National Laboratory) and sequenced. A full
length cDNA sequence was isolated for human ICACC-1 and 2 by
library screening and 5'-and 3' RACE cloning (Clonetech). Analysis
of the encoded murine protein sequence identified several conserved
motifs including multiple transmembrane domains and several
phosphorylation and glycosylation sites.
[0055] Expression of murine ICACC-1 was undetectable using standard
commercial tissue blots but elevated expression of ICACC-1 was
observed in lung, lymph node, colon, spleen, stomach, uterus and
ovary derived from IL-9 transgenic mice (FIG. 9). Interestingly,
these tissues all contain various epithelial cell types, suggesting
that this gene may be restricted to IL-9 responsive epithelial
cells. This data is supported by the finding that ICACC-1 gene
expression is induced in antigen exposed mice and this induction
can be suppressed by anti-IL9 treatment (FIGS. 10, 12, and 13).
[0056] To further understand which cell type is capable of
expressing ICACC-1, Applicants tested human lung epithelial cells
for their responsiveness to IL-9 as determined by ICACC-1 induced
gene expression. As shown in FIG. 14, the human lung epithelial
cell line designated NHBE (Clonetics), expressed ICACC-1 mRNA when
grown in the presence, but not in the absence of IL-9. When human
primary lung cultures were grown in the presence of recombinant
IL-9, ICACC-1 expression was induced in contrast to cell cultures
grown in medium alone (FIG. 15).
[0057] The nucleic acid molecules of the present invention include
nucleic acid molecules that encode the proteins having SEQ ID No.2,
SEQ ID No.4, SEQ ID NO: 6 and the related proteins herein
described, preferably in isolated form. As used herein, "nucleic
acid" is defined as RNA or DNA that encodes a protein or peptide as
defined above, or is complementary to nucleic acid sequence
encoding such peptides, or hybridizes to such nucleic acid and
remains stably bound to it under appropriate stringency conditions,
or encodes a polypeptide sharing at least 55% sequence identity,
75% sequence identity, preferably at least 80%, and more preferably
at least 85%, with the peptide sequences. Specifically contemplated
are genomic DNA, cDNA, mRNA and antisense molecules, as well as
nucleic acids based on alternative backbones or including
alternative bases whether derived from natural sources or
synthesized. Such hybridizing or complementary nucleic acids,
however, are defined further as being novel and unobvious over any
prior art nucleic acid including that which encodes, hybridizes
under appropriate stringency conditions, or is complementary to
nucleic acid encoding a protein according to the present
invention.
[0058] Homology or identity is determined by BLAST (Basic Local
Aligment Search Tool) analysis using the algorithm employed by the
programs blastp, blastn, blastx, tblastn and tblastx (Karlin, et
al. Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul,
S. F. J. Mol. Evol. 36: 290-300(1993), fully incorporated by
reference) which are tailored for sequence similarity searching.
The approach used by the BLAST program is to first consider similar
segments between a query sequence and a database sequence, then to
evaluate the statistical significance of all matches that are
identified and finally to summarize only those matches which
satisfy a preselected threshold of significance. For a discussion
of basic issues in similarity searching of sequence databases, see
Altschul et al. (Nature Genetics 6: 119-129 (1994)) which is fully
incorporated by reference. The search parameters for histogram,
descriptions, alignments, expect (i.e., the statistical
significance threshold for reporting matches against database
sequences), cutoff, matrix and filter are at the default settings.
The default scoring matrix used by blastp, blastx, tblastn, and
tblastx is the BLOSUM62 matrix (Henikoff, et al. Proc. Natl. Acad.
Sci. USA 89: 10915-10919 (1992), fully incorporated by reference).
For blastn, the scoring matrix is set by the ratios of M (i.e., the
reward score for a pair of matching residues) to N (i.e., the
penalty score for mismatching residues), wherein the default values
for M and N are 5 and -4, respectively.
[0059] "Stringent conditions" are those that (1) employ low ionic
strength and high temperature for washing, for example, 0.01 5M
NaCl/0.0015M sodium titrate/0.1% SDS at 50.degree. C., or (2)
employ during hybridization a denaturing agent such as formamide,
for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/0.1% FicolV0.1% polyvinylpyrrolidone/50 mM sodium phosphate
buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at
42.degree. C. Another example is use of 50% formamide, 5.times.SSC
(0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH
6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's solution,
sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran
sulfate at 42.degree. C., with washes at 42.degree. C. in
0.2.times.SSC and 0.1% SDS. A skilled artisan can readily determine
and vary the stringency conditions appropriately to obtain a clear
and detectable hybridization signal.
[0060] As used herein, a nucleic acid molecule is said to be
"isolated" when the nucleic acid molecule is substantially
separated from contaminant nucleic acid encoding other polypeptides
from the source of nucleic acid.
[0061] The present invention further provides fragments of the
encoding nucleic acid molecule. As used herein, a fragment of an
encoding nucleic acid molecule refers to a small portion of the
entire protein encoding sequence. The size of the fragment will be
determined by the intended use. For example, if the fragment is
chosen so as to encode an active portion of the protein, the
fragment will need to be large enough to encode the functional
region(s) of the protein or may encode regions of homology between
the ICACC proteins in FIG. 5. If the fragment is to be used as a
nucleic acid probe or PCR primer, then the fragment length is
chosen so as to obtain a relatively small number of false positives
during probing/priming.
[0062] Fragments of the encoding nucleic acid molecules of the
present invention (i.e., synthetic oligonucleotides) that are used
as probes or specific primers for the polymerase chain reaction
(PCR), or to synthesize gene sequences encoding proteins of the
invention can easily be synthesized by chemical techniques, for
example, the phosphotriester method of Matteucci, et al., (J. Am.
Chem. Soc. 103:3185-3191, 1981) or using automated synthesis
methods. In addition, larger DNA segments can readily be prepared
by well known methods, such as synthesis of a group of
oligonucleotides that define various modular segments of the gene,
followed by ligation of oligonucleotides to build the complete
modified gene.
[0063] The encoding nucleic acid molecules of the present invention
may further be modified so as to contain a detectable label for
diagnostic and probe purposes. A variety of such labels are known
in the art and can readily be employed with the encoding molecules
herein described. Suitable labels include, but are not limited to,
biotin, radiolabeled nucleotides and the like. A skilled artisan
can employ any of the art known labels to obtain a labeled encoding
nucleic acid molecule.
[0064] Modifications to the primary structure itself by deletion,
addition, or alteration of the amino acids incorporated into the
protein sequence during translation can be made without destroying
the activity of the protein. Such substitutions or other
alterations result in proteins having an amino acid sequence
encoded by a nucleic acid falling within the contemplated scope of
the present invention.
[0065] C. Isolation of Other Related Nucleic Acid Molecules
[0066] As described above, the identification of the murine nucleic
acid molecule having SEQ ID NO: 1 and the human nucleic acid
molecules having SEQ ID No.3 or SEQ ID No 5 allows a skilled
artisan to isolate nucleic acid molecules that encode other members
of the ICACC protein family in addition to the murine or human
sequences herein described.
[0067] Essentially, a skilled artisan can readily use the amino
acid sequence of SEQ ID NOS: 2, 4 or 6 to generate antibody probes
to screen expression libraries prepared from appropriate cells.
Typically, polyclonal antiserum from mammals such as rabbits
immunized with the purified protein (as described below) or
monoclonal antibodies can be used to probe a mammalian cDNA or
genomic expression library, such as lambda gtll library, to obtain
the appropriate coding sequence for other members of the protein
family. The cloned cDNA sequence can be expressed as a fusion
protein, expressed directly using its own control sequences, or
expressed by constructions using control sequences appropriate to
the particular host used for expression of the enzyme.
[0068] Alternatively, a portion of the coding sequence herein
described can be synthesized and used as a probe to retrieve DNA
encoding a member of the protein family from any mammalian
organism. Oligomers containing approximately 18-20 nucleotides
(encoding about a 6-7 amino acid stretch) are prepared and used to
screen genomic DNA or cDNA libraries to obtain hybridization under
stringent conditions or conditions of sufficient stringency to
eliminate an undue level of false positives.
[0069] Additionally, pairs of oligonucleotide primers can be
prepared for use in a polymerase chain reaction (PCR) to
selectively clone an encoding nucleic acid molecule. A PCR
denature/anneal/extend cycle for using such PCR primers is well
known in the art and can readily be adapted for use in isolating
other encoding nucleic acid molecules.
[0070] D. rDNA Molecules Containing a Nucleic Acid Molecule
[0071] The present invention further provides recombinant DNA
molecules (rDNAs) that contain a ICACC coding sequence. As used
herein, a rDNA molecule is a DNA molecule that has been subjected
to molecular manipulation in situ. Methods for generating rDNA
molecules are well known in the art, for example, see Sambrook et
al., Molecular Cloning (1989). In the preferred rDNA molecules, a
coding DNA sequence is operably linked to expression control
sequences and/or vector sequences.
[0072] The choice of vector and/or expression control sequences to
which one of the protein family encoding sequences of the present
invention is operably linked depends directly, as is well known in
the art, on the functional properties desired, e.g., protein
expression, and the host cell to be transformed. A vector
contemplated by the present invention is at least capable of
directing the replication or insertion into the host chromosome,
and preferably also expression, of the structural gene included in
the rDNA molecule.
[0073] Expression control elements that are used for regulating the
expression of an operably linked protein encoding sequence are
known in the art and include, but are not limited to, inducible
promoters, constitutive promoters, secretion signals, and other
regulatory elements. Preferably, the inducible promoter is readily
controlled, such as being responsive to a nutrient in the host
cell's medium.
[0074] In one embodiment, the vector containing a coding nucleic
acid molecule will include a prokaryotic replicon, i.e., a DNA
sequence having the ability to direct autonomous replication and
maintenance of the recombinant DNA molecule extrachromosomally in a
prokaryotic host cell, such as a bacterial host cell, transformed
therewith. Such replicons are well known in the art. In addition,
vectors that include a prokaryotic replicon may also include a gene
whose expression confers a detectable marker such as a drug
resistance. Typical bacterial drug resistance genes are those that
confer resistance to ampicillin or tetracycline.
[0075] Vectors that include a prokaryotic replicon can further
include a prokaryotic or bacteriophage promoter capable of
directing the expression (transcription and translation) of the
coding gene sequences in a bacterial host cell, such as E. coli. A
promoter is an expression control element formed by a DNA sequence
that permits binding of RNA polymerase and transcription to occur.
Promoter sequences compatible with bacterial hosts are typically
provided in plasmid vectors containing convenient restriction sites
for insertion of a DNA segment of the present invention. Typical of
such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available
from Biorad Laboratories, (Richmond, Calif.), pPL and pKK223
available from Pharmacia, Piscataway, N.J.
[0076] Expression vectors compatible with eukaryotic cells,
preferably those compatible with vertebrate cells, can also be used
to form a rDNA molecules that contains a coding sequence.
Eukaryotic cell expression vectors are well known in the art and
are available from several commercial sources. Typically, such
vectors are provided containing convenient restriction sites for
insertion of the desired DNA segment Typical of such vectors are
pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International
Biotechnologies, Inc.), pTDT1 (ATCC, #31255), the vector pCDM8
described herein, and the like eukaryotic expression vectors.
[0077] Eukaryotic cell expression vectors used to construct the
rDNA molecules of the present invention may further include a
selectable marker that is effective in an eukaryotic cell,
preferably a drug resistance selection marker. A preferred drug
resistance marker is the gene whose expression results in neomycin
resistance, i.e., the neomycin phosphotransferase (neo) gene.
(Southern et al., J. Mol. Anal. Genet. 1:327-341, 1982.)
Alternatively, the selectable marker can be present on a separate
plasmid, and the two vectors are introduced by co-transfection of
the host cell, and selected by culturing in the appropriate drug
for the selectable marker.
[0078] E. Host Cells Containing an Exogenously Supplied Coding
Nucleic Acid Molecule
[0079] The present invention further provides host cells
transformed with a nucleic acid molecule that encodes an ICACC
protein, preferably an ICACC-1 protein, of the present invention.
The host cell can be either prokaryotic or eukaryotic. Eukaryotic
cells useful for expression of a protein of the invention are not
limited, so long as the cell line is compatible with cell culture
methods and compatible with the propagation of the expression
vector and expression of the gene product. Preferred eukaryotic
host cells include, but are not limited to, yeast, insect and
mammalian cells, preferably vertebrate cells such as those from a
mouse, rat, monkey or human cell line. Preferred eukaryotic host
cells include Chinese hamster ovary (CHO) cells available from the
ATCC as CCL61, NIH Swiss mouse embryo cells NH/3T3 available from
the ATCC as CRL 1658, baby hamster kidney cells (13HK), and the
like eukaryotic tissue culture cell lines.
[0080] Any prokaryotic host can be used to express a rDNA molecule
encoding a protein of the invention. The preferred prokaryotic host
is E. coli.
[0081] Transformation of appropriate cell hosts with a rDNA
molecule of the present invention is accomplished by well known
methods that typically depend on the type of vector used and host
system employed. With regard to transformation of prokaryotic host
cells, electroporation and salt treatment methods are typically
employed, see, for example, Cohen et al., Proc. Natl. Acad. Sci.
USA 69:2110, 1972; and Maniatis et al., Molecular Cloning. A
Laboratory Mammal. Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1982). With regard to transformation of vertebrate
cells with vectors containing rDNAs, electroporation, cationic
lipid or salt treatment methods are typically employed, see, for
example, Graham et al., Virol. 52:456, 1973; Wigler et al., Proc.
Natl. Acad. Sci. USA 76:1373-76, 1979.
[0082] Successfully transformed cells, i.e., cells that contain a
rDNA molecule of the present invention, can be identified by well
known techniques including the selection for a selectable marker.
For example, cells resulting from the introduction of an rDNA of
the present invention can be cloned to produce single colonies.
Cells from those colonies can be harvested, lysed and their DNA
content examined for the presence of the rDNA using a method such
as that described by Southern, J. Mol. Biol. 98:503, 1975, or
Berent et al., Biotech. 3:208, 1985 or the proteins produced from
the cell assayed via an immunological method.
[0083] F. Production of Recombinant Proteins using a rDNA
Molecule
[0084] The present invention further provides methods for producing
an ICACC protein of the invention using nucleic acid molecules
herein described. In general terms, the production of a recombinant
form of a protein typically involves the following steps:
[0085] First, a nucleic acid molecule is obtained that encodes a
protein of the invention, such as the nucleic acid molecule
depicted in SEQ ID NOS. 1, 3 or 5 or the open reading frames of
these molecules. If the encoding sequence is uninterrupted by
introns, it is directly suitable for expression in any host.
[0086] The nucleic acid molecule is then preferably placed in
operable linkage with suitable control sequences, as described
above, to form an expression unit containing the protein open
reading frame. The expression unit is used to transform a suitable
host and the transformed host is cultured under conditions that
allow the production of the recombinant protein. Optionally the
recombinant protein is isolated from the medium or from the cells;
recovery and purification of the protein may not be necessary in
some instances where some impurities may be tolerated.
[0087] Each of the foregoing steps can be done in a variety of
ways. For example, the desired coding sequences may be obtained
from genomic fragments and used directly in appropriate hosts. The
construction of expression vectors that are operable in a variety
of hosts is accomplished using appropriate replicons and control
sequences, as set forth above. The control sequences, expression
vectors, and transformation methods are dependent on the type of
host cell used to express the gene and were discussed in detail
earlier. Suitable restriction sites can, if not normally available,
be added to the ends of the coding sequence so as to provide an
excisable gene to insert into these vectors. A skilled artisan can
readily adapt any host/expression system known in the art for use
with the nucleic acid molecules of the invention to produce
recombinant protein.
[0088] G. Methods to Identify Binding Partners
[0089] Another embodiment of the present invention provides methods
for use in isolating and identifying binding partners of proteins
of the invention In detail, a protein of the invention is mixed
with a potential binding partner or an extract or fraction of a
cell under conditions that allow the association of potential
binding partners with the protein of the invention. After mixing,
peptides, polypeptides, proteins or other molecules that have
become associated with a protein of the invention are separated
from the mixture. The binding partner that bound to the protein of
the invention can then be removed and further analyzed. To identify
and isolate a binding partner, the entire protein, for instance a
ICACC protein of SEQ D No.2, SEQ ID No. 4 or SEQ ID NO: 6 can be
used. Alternatively, a fragment of the protein or a membrane
fragment containing the protein may be used.
[0090] As used herein, a cellular extract refers to a preparation
or fraction which is made from a lysed or disrupted cell. The
preferred source of cellular extracts are cells derived from human
tissues or cells.
[0091] A variety of methods can be used to obtain an extract of a
cell. Cells can be disrupted using either physical or chemical
disruption methods. Examples of physical disruption methods
include, but are not limited to, sonication and mechanical
shearing. Examples of chemical lysis methods include, but are not
limited to, detergent lysis and enzyme lysis. A skilled artisan can
readily adapt methods for preparing cellular extracts in order to
obtain extracts for use in the present methods.
[0092] Once an extract of a cell is prepared, the extract is mixed
with the protein of the invention under conditions in which
association of the protein with the binding partner can occur. A
variety of conditions can be used, the most preferred being
conditions that closely resemble conditions found in the cytoplasm
of a human cell. Features such as osmolarity, pH, temperature, and
the concentration of cellular extract used, can be varied to
optimize the association of the protein with the binding
partner.
[0093] After mixing under appropriate conditions, the bound complex
is separated from the mixture. A variety of techniques can be
utilized to separate the mixture. For example, antibodies specific
to a protein of the invention can be used to immunoprecipitate the
binding partner complex. Alternatively, standard chemical
separation techniques such as chromatography and density/sediment
centrifugation can be used.
[0094] After removal of non-associated cellular constituents found
in the extract, the binding partner can be dissociated from the
complex using conventional methods. For example, dissociation can
be accomplished by altering the salt concentration or pH of the
mixture. To aid in separating associated binding partner pairs from
the mixed extract, the protein of the invention can be immobilized
on a solid support For example, the protein can be attached to a
nitrocellulose matrix or acrylic beads. Attachment of the protein
to a solid support aids in separating peptide/binding partner pairs
from other constituents found in the extract. The identified
binding partners can be either a single protein or a complex made
up of two or more proteins. Alternatively, binding partners may be
identified using a Far-Western assay according to the procedures of
Takayama et al. (1997) Methods Mol. Biol. 69:171-84 or Sauder et
al. J Gen.Virol. 77(5):991-6 or identified through the use of
epitope tagged proteins or GST fusion proteins.
[0095] Alternatively, the nucleic acid molecules of the invention
can be used in a yeast two-hybrid system, preferably systems for
screening binding partners of membrane proteins. The yeast
two-hybrid system has been used to identify other protein partner
pairs and can readily be adapted to employ the nucleic acid
molecules herein described.
[0096] H. Methods to Identify Agents that Modulate the Expression a
Nucleic Acid Encoding and ICACC Protein.
[0097] Another embodiment of the present invention provides methods
for identifying agents that modulate the expression of a nucleic
acid encoding a protein of the invention such as a protein having
the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6. Such assays
may utilize any available means of monitoring for changes in the
expression level of the nucleic acids of the invention. As used
herein, an agent is said to modulate the expression of a nucleic
acid of the invention, for instance a nucleic acid encoding the
protein having the sequence of SEQ ID NO:2 or SEQ ID NO:6 if it is
capable of up- or down-regulating expression of the nucleic acid in
a cell.
[0098] Agents of the invention may relate to antisense or gene
therapy. It is now known in the art that altered DNA molecules can
be tailored to provide a selected effect, when provided as
antisense or gene therapy. The native DNA segment coding for
ICACC-1 has two strands; a sense strand and an antisense strand
held together by hydrogen bonds. The mRNA coding for the receptor
has a nucleotide sequence identical to the sense strand, with the
expected substitution of thymidine by uridine. Thus, based upon the
knowledge of the receptor sequence, synthetic oligonucleotides can
be synthesized These oligonucleotides can bind to the DNA and RNA
coding for ICACC-1. The active fragments of the invention, which
are complementary to mRNA and the coding strand of DNA, are usually
at least about 15 nucleotides, more usually at least 20
nucleotides, preferably 30 nucleotides and more preferably may be
50 nucleotides or more. The binding strength between the sense and
antisense strands is dependent upon the total hydrogen bonds.
Therefore, based upon the total number of bases in the mRNA, the
optimal length of the oligonucleotide sequence may be easily
calculated by the skilled artisan.
[0099] The sequence may be complementary to any portion of the
sequence of the mRNA. For example, it may be proximal to the
5'-terminus or capping site or downstream from the capping site,
between the capping site and the initiation codon and may cover all
or only a portion of the non-coding region or the coding region.
The particular site(s) to which the antisense sequence binds will
vary depending upon the degree of inhibition desired, the
uniqueness of the sequence, the stability of the antisense
sequence, etc.
[0100] In the practice of the invention, expression of ICACC-1 is
down-regulated by administering an effective amount of antisense
oligonucleotide sequences described above. The oligonucleotide
compounds of the invention bind to the mRNA coding for human
ICACC-1 thereby inhibiting expression (translation) of these
proteins. The isolated DNA sequences, containing various mutations
such as point mutations, insertions, deletions or spliced mutations
of ICACC-1 are useful in gene therapy as well.
[0101] In one assay format for agents, cell lines that contain
reporter gene fusions between the open reading frame and any
assayable fusion partner may be prepared. Numerous assayable fusion
partners are known and readily available including the firefly
luciferase gene and the gene encoding chloramphenicol
acetyltransferase (Alam et al. (1990) Anal. Biochem. 188:245-254).
Cell lines containing the reporter gene fusions are then exposed to
the agent to be tested under appropriate conditions and time.
Differential expression of the reporter gene between samples
exposed to the agent and control samples identifies agents which
modulate the expression of a nucleic acid encoding an ICACC-1
protein.
[0102] Additional assay formats may be used to monitor the ability
of the agent to modulate the expression of a nucleic acid encoding
a protein of the invention, such as the protein having SEQ ID NO:2
or SEQ ID NO:6. For instance, mRNA expression may be monitored
directly by hybridization to the nucleic acids of the invention.
Cell lines are exposed to the agent to be tested under appropriate
conditions and time and total RNA or mRNA is isolated by standard
procedures such those disclosed in Sambrook et al. (Molecular
Cloning: A Laboratory Manual, 2nd Ed. Clod Spring Harbor Laboratory
Press, 1989). Probes to detect differences in RNA expression levels
between cells exposed to the agent and control cells may be
prepared from the nucleic acids of the invention. It is preferable,
but not necessary, to design probes which hybridize only with
target nucleic acids under conditions of high stringency. Only
highly complementary nucleic acid hybrids form under conditions of
high stringency. Accordingly, the stringency of the assay
conditions determines the amount of complementarity which should
exist between two nucleic acid strands in order to form a hybrid.
Stringency should be chosen to maximize the difference in stability
between the probe:target hybrid and potential probe:non-target
hybrids.
[0103] Probes may be designed from the nucleic acids of the
invention through methods known in the art. For instance, the G+C
content of the probe and the probe length can affect probe binding
to its target sequence. Methods to optimize probe specificity are
commonly available in Sambrook et al. (Molecular Cloning: A
Laboratory Approach, Cold Spring Harbor Press, NY, 1989) or Ausubel
et al. (Current Protocols in Molecular Biology, Greene Publishing
Co., NY, 1995).
[0104] Hybridization conditions are modified using known methods,
such as those described by Sambrook et al. and Ausubel et al. as
required for each probe. Hybridization of total cellular RNA or RNA
enriched for polyA RNA can be accomplished in any available format
For instance, total cellular RNA or RNA enriched for polyA RNA can
be affixed to a solid support and the solid support exposed to at
least one probe comprising at least one, or part of one of the
sequences of the invention under conditions in which the probe will
specifically hybridize. Alternatively, nucleic acid fragments
comprising at least one, or part of one of the sequences of the
invention can be affixed to a solid support, such as a porous glass
wafer. The glass wafer can then be exposed to total cellular RNA or
polyA RNA from a sample under conditions in which the affixed
sequences will specifically hybridize. Such glass wafers and
hybridization methods are widely available, for example, those
disclosed by Beattie (WO 95/11755). By examining for the ability of
a given probe to specifically hybridize to an RNA sample from an
untreated cell population and from a cell population exposed to the
agent, agents which up or down regulate the expression of a nucleic
acid encoding an ICACC protein, preferably an ICACC-1 protein, are
identified.
[0105] Hybridization for qualitative and quantitative analysis of
mRNAs may also be carried out by using a RNase Protection Assay
(i.e., RPA, see Ma et al. (1996) Methods 10: 273-238).
[0106] Briefly, an expression vehicle comprising cDNA encoding the
gene product and a phage specific DNA dependent RNA polymerase
promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the
3' end of the cDNA molecule, downstream from the phage promoter,
wherein such a linearized molecule is subsequently used as a
template for synthesis of a labeled antisense transcript of the
cDNA by in vitro transcription. The labeled transcript is then
hybridized to a mixture of isolated RNA (i.e., total or
fractionated mRNA) by incubation at 45.degree. C. overnight in a
buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl
and 1 mM EDTA. The resulting hybrids are then digested in a buffer
comprising 40 .mu.g/ml ribonuclease A and 2 .mu.g/ml ribonuclease.
After deactivation and extraction of extraneous proteins, the
samples are loaded onto urea/polyacrylamide gels for analysis.
[0107] In another assay format for agents which effect the
expression of the instant gene products, cells or cell lines would
first be identified which express said gene products
physiologically (e.g., see the Figures for tissue distribution).
Cell and/or cell lines so identified would be expected to comprise
the necessary cellular machinery such that the fidelity of
modulation of the transcriptional apparatus is maintained with
regard to exogenous contact of agent with appropriate surface
transduction mechanisms and/or the cytosolic cascades. Further,
such cells or cell lines would be transduced or transfected with an
expression vehicle (e.g., a plasmid or viral vector) construct
comprising an operable non-translated 5'-promoter containing end of
the structural gene encoding the insant gene products fused to one
or more antigenic fragments, which are peculiar to the instant gene
products, wherein said fragments are under the transcriptional
control of said promoter and are expressed as polypeptides whose
molecular weight can be distinguished from the naturally occurring
polypeptides or may further comprise an immunologically distinct
tag. Such a process is well known in the art (see Maniatis). Cells
may be exposed to IL-9.
[0108] Cells or cell lines transduced or transfected as outlined
above would then be contacted with agents under appropriate
conditions; for example, the agent comprises a pharmaceutically
acceptable excipient and is contacted with cells comprised in an
aqueous physiological buffer such as phosphate buffered saline
(PBS) at physiological pH, Eagles balanced salt solution (BSS) at
physiological pH, PBS or BSS comprising serum or conditioned media
comprising PBS or BSS and/or serum incubated at 37.degree. C. Said
conditions may be modulated as deemed necessary by one of skill in
the art Subsequent to contacting the cells with the agent, said
cells will be disrupted and the polypeptides of the disruptate are
fractionated such that a polypeptide fraction is pooled and
contacted with an antibody to be further processed by immunological
assay (e.g., ELISA, immunoprecipitation or Western blot). The pool
of proteins isolated from the "agent contacted" sample will be
compared with a control sample where only the excipient is
contacted with the cells and an increase or decrease in the
immunologically generated signal from the "agent contacted" sample
compared to the control will be used to distinguish the
effectiveness of the agent.
[0109] I. Methods to Identify Agents that Modulate at Least One
Activity of an ICACC Protein.
[0110] Another embodiment of the present invention provides methods
for identifying agents that modulate at least one activity of a
protein of the invention, such as a protein having the amino acid
sequence of SEQ ID No.2, SEQ ID NO:4 or SEQ ID No.6 and preferably,
an ICACC-1 protein. Such methods or assays may utilize any means of
monitoring or detecting the desired activity.
[0111] Specific assays may be based on monitoring the cellular
functions of ICACC-1. Antagonists of the invention include those
molecules that interact or bind to ICACC-1 and inactivate this
receptor. To identify other allosteric, inverse or weak antagonists
of the invention, one may test for binding to ICACC-1. The present
invention includes antagonists of ICACC-1 that block activation of
this receptor. Antagonists are compounds that are themselves devoid
of pharmacological activity but cause effects by preventing the
action of an agonist. To identify an antagonist of the invention,
one may test for competitive binding with natural ligands of
ICACC-1. Assays of antagonistic binding and activity can be derived
from monitoring ICACC-1 functions for down-regulation as described
herein and in the cited literature. The binding of the antagonist
may involve all known types of interactions including ionic forces,
hydrogen bonding, hydrophobic interactions, van der Waals forces
and covalent bonds. In many cases, bonds of multiple types are
important in the interaction of an agonist or antagonist with a
molecule like ICACC-1.
[0112] In a further embodiment, these compounds may be analogues of
ICACC-1 or its ligands. ICACC-1 analogues may be produced by point
mutations in the isolated DNA sequence for the gene, nucleotide
substitutions and/or deletions which can be created by methods that
are all well described in the art (Simoncsits et al., 1994). This
invention also includes spliced variants of ICACC-1 and discloses
isolated nucleic acid sequences of ICACC-1, which contain deletions
of one or more of its exons. The term "spliced variants" as used
herein denotes a purified and isolated DNA molecule encoding human
ICACC-1 comprising at least one exon. There is no evidence of
naturally expressed spliced mutants in the art. It must be
understood that these exons may contain various point
mutations.
[0113] Structure-activity relationships may be used to modify the
antagonists of the invention. For example, the techniques of X-ray
crystallography and NMR may be used to make modifications of the
invention. For example, one can create a three-dimensional
structure of human ICACC-1 that can be used as a template for
building structural models of deletion mutants using molecular
graphics. These models can then be used to identify and construct a
ligand for ICACC-1 which alters normal chloride channel function.
In still another embodiment, these compounds may also be used as
dynamic probes for ICACC-1 structure and to develop ICACC-1
antagonists using cell lines or other suitable means of assaying
ICACC-1 activity.
[0114] In addition, this invention also provides compounds that
prevent the synthesis or reduce the biologic stability of ICACC-1.
Biologic stability is a measure of the time between the synthesis
of the molecule and its degradation. For example, the stability of
a protein, peptide or peptide mimetic (Kauvar, 1996) therapeutic
may be prolonged by using D-amino acids or shortened by altering
its sequence to make it more susceptible to enzymatic
degradation.
[0115] In another embodiment, antagonists of the invention are
antibodies to ICACC-1. The antibodies to ICACC-1 may be either
monoclonal or polyclonal, made using standard techniques well known
in the art (See Harlow & Lane's Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1988). They can be
used to block ICACC-1 activation by binding to extracellular
regions of the protein required for ligand binding or activation.
In one embodiment, the antibodies interact with ICACC-1, in another
they interact with the ligands for ICACC-1. The ICACC-1 used to
elicit these antibodies can be the ICACC-1 protein or any of the
ICACC-1 variants or fragments discussed above. Antibodies are also
produced from peptide sequences of ICACC-1 using standard
techniques in the art (see Protocols in Immunology," Jobn Wiley
& Sons, 1994).
[0116] In one assay format, the relative amounts of ICACC-1 protein
of the invention between a cell population that has been exposed to
the agent to be tested compared to an unexposed control cell
population may be assayed. In this format, probes such as specific
antibodies are used to monitor the differential expression of the
protein in the different cell populations. Cell lines or
populations are exposed to the agent to be tested under appropriate
conditions and time. Cellular lysates may be prepared from the
exposed cell line or population and a control, unexposed cell line
or population. The cellular lysates are then analyzed with the
probe.
[0117] Antibody probes are prepared by immunizing suitable
mammalian hosts in appropriate immunization protocols using the
peptides. polypeptides or proteins of the invention if they are of
sufficient length, or, if desired, or if required to enhance
immunogenicity, conjugated to suitable carriers. Methods for
preparing immunogenic conjugates with carriers such as BSA, KLH, or
other carrier proteins are well known in the art. In some
circumstances, direct conjugation using, for example, carbodiimide
reagents may be effective; in other instances linking reagents such
as those supplied by Pierce Chemical Co., Rockford, Ill., may be
desirable to provide accessibility to the hapten. The hapten
peptides can be extended at either the amino or carboxy terminus
with a Cys residue or interspersed with cysteine residues, for
example, to facilitate linking to a carrier. Administration of the
immunogens is conducted generally by injection over a suitable time
period and with use of suitable adjuvants, as is generally
understood in the art. During the immunization schedule, titers of
antibodies are taken to determine adequacy of antibody
formation.
[0118] While the polyclonal antisera produced in this way may be
satisfactory for some applications, for pharmaceutical
compositions, use of monoclonal preparations is preferred.
Immortalized cell lines which secrete the desired monoclonal
antibodies may be prepared using the standard method of Kohler and
Milstein or modifications which effect immortalization of
lymphocytes or spleen cells, as is generally known. The
immortalized cell lines secreting the desired antibodies are
screened by immunoassay in which the antigen is the peptide hapten,
polypeptide or protein. When the appropriate immortalized cell
culture secreting the desired antibody is identified, the cells can
be cultured either in vitro or by production in ascites fluid.
[0119] The desired monoclonal antibodies are then recovered from
the culture supernatant or from the ascites supernatant. Fragments
of the monoclonals or the polyclonal antisera which contain the
immunologically significant portion can be used as antagonists, as
well as the intact antibodies. Use of immunologically reactive
fragments, such as the Fab, Fab', of F(ab').sub.2 fragments is
often preferable, especially in a therapeutic context, as these
fragments are generally less immunogenic than the whole
immunoglobulin.
[0120] The antibodies or fragments may also be produced, using
current technology, by recombinant means. Antibody regions that
bind specifically to the desired regions of the protein can also be
produced in the context of chimeras with multiple species origin.
Agents that are assayed in the above method can be randomly
selected or rationally selected or designed. As used herein, an
agent is said to be randomly selected when the agent is chosen
randomly without considering the specific sequences involved in the
association of the a protein of the invention alone or with its
associated substrates, binding partners, etc. An example of
randomly selected agents is the use a chemical library or a peptide
combinatorial library, or a growth broth of an organism.
[0121] As used herein, an agent is said to be rationally selected
or designed when the agent is chosen on a nonrandom basis which
takes into account the sequence of the target site and/or its
conformation in connection with the agent's action.
[0122] The agents of the present invention can be, as examples,
peptides, small molecules, vitamin derivatives, as well as
carbohydrates. A skilled artisan can readily recognize that there
is no limit as to the structural nature of the agents of the
present invention. The peptide agents of the invention can be
prepared using standard solid phase (or solution phase) peptide
synthesis methods, as is known in the art. In addition, the DNA
encoding these peptides may be synthesized using commercially
available oligonucleotide synthesis instrumentation and produced
recombinantly using standard recombinant production systems. The
production using solid phase peptide synthesis is necessitated if
non-gene-encoded amino acids are to be included.
[0123] Another class of agents of the present invention are
antibodies immunoreactive with critical positions of proteins of
the invention. Antibody agents are obtained by immunization of
suitable mammalian subjects with peptides, containing as antigenic
regions, those portions of the protein intended to be targeted by
the antibodies.
[0124] J. Uses for Agents that Modulate at Least One Activity of an
ICACC Protein.
[0125] Further evidence defining the role of ICACC-1 in the
pathogenesis of atopic allergy, bronchial hyperresponsiveness,
asthma and related disorders is derived directly from the
Applicants observation that IL-9 selectively induces ICACC-1. Thus,
the pleiotropic role for IL-9, which is important to a number of
antigen induced responses is dependent in part, on the
up-regulation of ICACC-1 in cells critical to atopic allergy. When
the functions of IL-9 are down-regulated by antibody pretreatment
prior to aerosol challenge with antigen, animals can be completely
protected from the antigen induced responses. These responses
include: bronchial hyperresponsiveness, eosinophilia and elevated
cell counts in bronchial lavage, histologic changes in lung
associated with inflammation and elevated serum IgE. The
suppression of IL 9 and asthmatic-like responses is associated with
down regulated expression of ICACC-1 (FIG. 13). Thus, treatment of
such responses, which underlie the pathogenesis of atopic allergy
and characterize allergic inflammation associated with this
disorder, by down-regulating ICACC-1, is within the scope of this
invention.
[0126] The involvement of chloride channels in IL-9 biologic
responses is addressed by in vitro primary lung cultures that
produce secreted eotaxin protein upon IL-9 stimulation (FIG. 17).
The treatment of these cultures with known chloride channel
inhibitors results in suppression of the IL-9 induced eotaxin
response (FIG. 18) and thus provides an assay for screening for
ICACC-1 inhibitors. In another embodiment cell lines in which
ICACC-1 expression vectors are introduced can be used to screen for
specific chloride channel inhibitors.
[0127] Applicants also teach the down-regulation of ICACC-1 by
administering antagonists of ICACC-1. The skilled artisan will
recognize that all molecules containing the requisite
three-dimensional structural conformation critical for activation
of, or ligand binding to ICACC-1 are within the scope of this
invention.
[0128] The demonstration of an IL-9 sequence associated with an
asthma-like phenotype and one associated with the absence of an
asthma-like phenotype, indicates that the inflammatory response to
antigen in the lung is IL-9 dependent Down-regulating ICACC-1,
which is selectively induced downstream in the IL-9 pathway, will
therefore protect against this antigen induced response.
[0129] In addition to the direct regulation of the ICACC-1 gene,
this invention also encompasses methods of inhibiting the
intracellular signaling by ICACC-1. It is known in the art that
highly exergonic phosphoryl-transfer reactions are catalyzed by
various enzymes known as kinases. In other words, a kinase
transfers phosphoryl groups between ATP and a metabolite. Included
within the scope of this invention are specific inhibitors of
protein kinases. Thus, inhibitors of these kinases are useful in
the down-regulation of ICACC-1 and are therefore useful in the
treatment of atopic allergies and asthma
[0130] In still another aspect of the invention, surprisingly,
aminosterol compounds were found to be useful in the inhibition of
ICACC-1 induction by IL-9. Aminosterol compounds which are useful
in this invention are described in U.S. patent application Ser. No.
08/290,826 and its related application Ser. Nos. 08/416,883 and
08/478,763 as well as in Ser. No. 08/483,059 and its related
application Ser. Nos. 08/483,057, 08/479,455, 08/479,457,
08/475,572, 08/476,855, 08/474,799 and 08/487,443, which are
specifically incorporated herein by reference in their
entirety.
[0131] While a therapeutic potential for ICACC-1 down-regulation
has been identified, Applicants have also recognized a therapeutic
potential for up-regulation of ICACC-1 as well. Patients with
cystic fibrosis are hampered by lung disease characterized by thick
secretions, which cause airway obstruction and subsequent
colonization and infection by inhaled pathogenic microorganisms
(Eng et al., 1996). Airway epithelia from cystic fibrosis patients
exhibit a broad spectrum of ion transport properties that differ
from normal, including not only defective cAMP-mediated chloride
secretion, but also increased sodium absorption and increased
calcium-mediated chloride secretion (Johnson et al., 1995).
Restoration of overall chloride secretion in primary cystic
fibrosis airway epithelial cells leads to correction of sodium
hyperabsorption and normal airway epithelial cell function (Johnson
et al., 1995). Applicants therefore provide a method for treating
cystic fibrosis by further increasing calcium-dependent chloride
secretion in these cells through up-regulation of ICACC-1 activity
in airway epithelia In this manner, the decrease in chloride
secretion due to the defect in cAMP-mediated chloride secretion is
compensated for through up-regulation of ICACC-1. The result being
a restoration of the cellular chloride gradient and normal airway
epithelial cell function. In another indication, up regulation of
ICACC-1 will be useful for treating autoimmune associated diseases
such as IBD.
[0132] As provided in the Examples, the proteins and nucleic acids
of the invention, such as the ICACC-1 proteins having the amino
acid sequence of SEQ ID NOS: 2 or 6, are induced by IL-9. Agents
that modulate or down-regulate the expression of the protein or
agents such as agonists or antagonists of at least one activity of
the protein may be used to modulate biological and pathologic
processes associated with the protein's function and activity. As
used herein, a subject can be any mammal, so long as the mammal is
in need of modulation of a pathological or biological process
mediated by a protein of the invention.
[0133] The term "mammal" is meant an individual belonging to the
class Mammalia. The invention is particularly useful in the
treatment of human subjects.
[0134] Pathological processes refer to a category of biological
processes which produce a deleterious effect. For example,
expression of a protein of the invention may be associated with
atopic allergy, asthma and/or cystic fibrosis. As used herein, an
agent is said to modulate a pathological process when the agent
reduces the degree or severity of the process. For instance, atopic
allergy, asthma and/or cystic fibrosis may be prevented or disease
progression modulated by the administration of agents which reduce
or modulate in some way the expression or at least one activity of
a protein of the invention.
[0135] The agents of the present invention can be provided alone,
or in combination with other agents that modulate a particular
pathological process. For example, an agent of the present
invention can be administered in combination with anti-asthma
agents. As used herein, two agents are said to be administered in
combination when the two agents are administered simultaneously or
are administered independently in a fashion such that the agents
will act at the same time.
[0136] The agents of the present invention can be administered via
parenteral, subcutaneous, intravenous, intramuscular,
intraperitoneal, transdermal, or buccal routes. Alternatively, or
concurrently, administration may be by the oral route or directly
to the lungs. The dosage administered will be dependent upon the
age, health, and weight of the recipient, kind of concurrent
treatment, if any, frequency of treatment, and the nature of the
effect desired.
[0137] The compounds used in the method of treatment of this
invention may be administered systemically or topically, depending
on such considerations as the condition to be treated, need for
site-specific treatment, quantity of drug to be administered and
similar considerations.
[0138] Topical administration may be used. Any common topical
formation such as a solution, suspension, gel, ointment or salve
and the like may be employed Preparation of such topical
formulations are well described in the art of pharmaceutical
formulations as exemplified, for example, by Remington's
Pharmaceutical Sciences. For topical application, these compounds
could also be administered as a powder or spray, particularly in
aerosol form. The active ingredient may be administered in
pharmaceutical compositions adapted for systemic administration. As
is known, if a drug is to be administered systemically, it may be
confected as a powder, pill, tablet or the like or as a syrup or
elixir for oral administration. For intravenous, intraperitoneal or
intra-lesional administration, the compound will be prepared as a
solution or suspension capable of being administered by injection.
In certain cases, it may be useful to formulate these compounds in
suppository form or as an extended release formulation for deposit
under the skin or intramuscular injection. In a preferred
embodiment, the compounds of this invention may be administered by
inhalation. For inhalation therapy the compound may be in a
solution useful for administration by metered dose inhalers or in a
form suitable for a dry powder inhaler.
[0139] An effective amount is that amount which will down-regulate
ICACC-1. A given effective amount will vary from condition to
condition and in certain instances may vary with the severity of
the condition being treated and the patient's susceptibility to
treatment. Accordingly, a given effective amount will be best
determined at the time and place through routine experimentation
However, it is anticipated that in the treatment of atopic allergy
and asthma-related disorders in accordance with the present
invention, a formulation containing between 0.001 and 5 percent by
weight, preferably about 0.01 to 1%, will usually constitute a
therapeutically effective amount When administered systemically, an
amount between 0.01 and 100 mg per kg body weight per day, but
preferably about 0.1 to 10 mg/kg, will effect a therapeutic result
in most instances.
[0140] The invention also includes pharmaceutical compositions
comprising the compounds of the invention together with a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil sesame oil and the like. Water
is a preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers are described in Remington's Pharmaceutical Sciences, Mack
Publishing Company, 1995. In addition to the pharmacologically
active agent, the compositions of the present invention may contain
suitable pharmaceutically acceptable carriers comprising excipients
and auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically for delivery
to the site of action. Suitable formulations for parenteral
administration include aqueous solutions of the active compounds in
water-soluble form, for example, water-soluble salts. In addition,
suspensions of the active compounds as appropriate oily injection
suspensions may be administered. Suitable lipophilic solvents or
vehicles include fatty oils, for example, sesame oil, or synthetic
fatty acid esters, for example, ethyl oleate or triglycerides.
Aqueous injection suspensions may contain substances which increase
the viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the
suspension may also contain stabilizers. Liposomes can also be used
to encapsulate the agent for delivery into the cell.
[0141] The pharmaceutical formulation for systemic administration
according to the invention may be formulated for enteral,
parenteral or topical administration Indeed, all three types of
formulations may be used simultaneously to achieve systemic
administration of the active ingredient.
[0142] Suitable formulations for oral administration include hard
or soft gelatin capsules, pills, tablets, including coated tablets,
elixirs, suspensions, syrups or inhalations and controlled release
forms thereof.
[0143] In practicing the methods of this invention, the compounds
of this invention may be used alone or in combination, or in
combination with other therapeutic or diagnostic agents. In certain
preferred embodiments, the compounds of this invention may be
coadministered along with other compounds typically prescribed for
these conditions according to generally accepted medical practice.
The compounds of this invention can be utilized in vivo, ordinarily
in mammals, preferably in humans.
[0144] In still another embodiment, the compounds of the invention
may be coupled to chemical moieties, including proteins that alter
the functions or regulation of ICACC-1 for therapeutic benefit in
atopic allergy and asthma (Kreitman et al., 1994). These proteins
may include in combination other inhibitors of cytokines and growth
factors including anti-IL-4, anti-IL-5, anti-L3, anti-IL-2,
anti-IL-13, anti-IL-11 and anti-IL-10 that may offer additional
therapeutic benefit in atopic allergy and asthma In addition, the
molecules of the invention may also be conjugated through
phosphorylation to biotinylate, thioate, acetylate, iodinate using
any of the cross-linking reagents well known in the art.
[0145] K. Diagnostics
[0146] Also included in the invention are methods of diagnosing
susceptibility to atopic allergy and related disorders and for
treating these disorders based on the relationship between IL 9,
its receptor and ICACC-1.
[0147] These disorders also include the monitoring of ICACC-1 gene
expression for the diagnosis of autoimmune disease of the bowel
such as inflammatory bowel disease (IBD). In the case of IBD the
lack or suppression of ICACC-1 gene expression would be a
diagnostic marker for the disease and the ability to follow ICACC-1
levels would aid in monitoring treatment.
[0148] One diagnostic embodiment involves the recognition of
variations in the DNA sequence of ICACC-1. One method involves the
introduction of a nucleic acid molecule (also known as a probe)
having a sequence complementary to ICACC-1 of the invention under
sufficient hybridizing conditions, as would be understood by those
in the art. In one embodiment, the sequence will bind specifically
to one allele of ICACC-1 or a fragment thereof and in another
embodiment will bind to both alleles. Another method of recognizing
DNA sequence variation associated with these disorders is direct
DNA sequence analysis by multiple methods well known in the art
(Ott, 1991). Another embodiment involves the detection of DNA
sequence variation in the ICACC-1 gene associated with these
disorders (Schwengel et al., 1993; Sheffield et al., 1993; Orita et
al., 1989; Sarkar et al., 1992; Cotton, 1989). These include the
polymerase chain reaction, restriction fragment length polymorphism
analysis and single stranded conformational analysis.
[0149] The practice of the present invention will employ the
conventional terms and techniques of molecular biology,
pharmacology, immunology and biochemistry that are within the
ordinary skill of those in the art. For example, see Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold
Spring Harbor Laboratory Press, 1985.
[0150] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
EXAMPLES
Example 1
cDNA Difference Analysis of IL-9 Expressed Genes
[0151] Lungs extracted from transgenic IL-9 mice (Tg5) were used to
isolate IL-9 induced genes. Tg5 is a FVB mouse overexpressing the
IL-9 gene as previously described (Renauld et al., 1994). This
strain has been shown to overexpress IL-9 in most tissues of the
mouse. In order to identify specific IL-9 induced genes,
suppressive PCR cDNA difference analysis was performed on mRNA from
lungs of Tg5 mice and parental FVB mice using a commercially
available PCR-select cDNA subtraction kit (Clonetech).
[0152] cDNA synthesis. Total RNA was prepared from lungs of FVB and
Tg5 mice using Trizol as described by the manufacturer (Gibco/BRL).
Lungs were removed from euthanized mice and frozen in liquid
nitrogen. Frozen lungs were then placed in Trizol and pulverized
using a tissue grinder. Polyadenylated RNA was purified from total
RNA with oligo(dT) cellulose columns (Pharmacia). Double stranded
cDNA was prepared using Superscript II reverse transcriptase and an
oligo(dT) primer as suggested by the manufacturer (Clonetech). cDNA
was then prepared by phenol-chloroform extraction and ethanol
precipitation. Products were resuspended in nuclease-free water and
analyzed on agarose gels to determine quality of products as
described below.
[0153] cDNA difference analysis protocol. Differential cDNA
analysis of Tg5 and FVB lungs was carried out following the
manufacturers protocol (Clonetech) as depicted in FIG. 1. The
results of the subtraction between the cDNA of these lungs resulted
in the generation of 1200 recombinant clones. Analysis of these
clones revealed multiples of several species, each accounting for
2-5% of the library. The most prominent transcript in the library
was the IL-9 cDNA which served as a control for the efficiency of
subtraction since it was a subtraction between an IL-9
constitutively expressing mouse (Tg5) and its parental control.
Another cDNA which was found in multiple copies (represented 3% of
library) was a novel calcium activated chloride channel which is
described below.
Example 2
Identification of the Murine ICACC-1 cDNA in the Lung of IL-9
Transgenic Mice
[0154] ICACC-1 probes as described in Example 1 were used to probe
a murine lung cDNA library (Clonetech) according to the
manufacturers recommendation. One million recombinant clones were
screened and several overlapping phage were identified. Subsequent
screens enabled identification and isolation of a single plaque
containing a phagemid which was then transformed into a
double-stranded plasmid by phage rescue according to the
manufacturers protocol. Recombinant clones were prepared and
sequenced using primers directed to the plasmid vector as well as
internal sequences identified from the partially subtracted probe.
Clones were then aligned and contiged to generate the full-length
sequence.
[0155] The 2931 bp cDNA isolated contained an open reading frame
encoding a protein of 925 amino acids. FIG. 2 shows the nucleotide
and amino acid sequence of the murine ICACC-1 cDNA. A nucleotide
BLAST (Altschul et al., 1990) database search of GenBank with the
full length cDNA revealed that it was similar to the bovine
chloride channel protein. FIG. 3 shows an alignment to the bovine
calcium activated chloride channel cDNA. Motif analysis of the
encoded polypeptide demonstrated several features such as multiple
transmembrane regions and glycosylation sites. The primary sequence
of murine ICACC-1 was used to perform an EST database search and
several undescribed human ESTs were found to be homologous to small
portions of the novel cDNA. FIGS. 4A and 4B show the sequences of
the human ICACC-1 and ICACC-2 genes. Both full length human ICACC
sequences were obtained by screening a human cDNA library.
Example 3
ICACC-1 is induced in vivo by IL-9 in Murine Cells
[0156] To confirm that ICACC-1 is induced by IL-9 in the lung, RNA
from the lungs of Tg5 and FVB mice were isolated as described in
Example 1. cDNA was generated using random hexamers (Phrmacia) and
Superscript II (Gibco/BRL) as suggested by the manufacturer.
Message was analyzed by PCR as described (Nicolaides et al., 1995)
and via Northern blot Primers used to generate murine ICACC-1
message were; sense 5'-CCAGATCCACACCAAAACGAGAA- G-3' (SEQ ID NO:7)
(nucleotides 689-712) and antisense 5'-CACTGTCAAAGGTCACCATCCCGA-3'
(SEQ ID NO:8) (nucleotides 1041-1064) which produce a gene product
of 376 bp. DHFR was assayed as an internal control to measure for
cDNA integrity using primers previously described (Nicolaides et
al, 1991). Amplification conditions used were 95.degree. C. for 30
seconds, 58.degree. C. for 1.5 minutes and 72.degree. C. for 1.5
minutes for 35 cycles. For Northern blot analysis, total RNA
derived from Tg5 or FVB lungs was electrophoresed on 1.5%
formaldehyde gels, transferred to nylon membranes and probed with a
DNA fragment comprising the murine ICACC-1 cDNA.
[0157] The results of the expression studies demonstrated that
ICACC-1 is specifically expressed in the lung of the IL-9
transgenic mouse but not in the parental strain (FIG. 6). This data
demonstrated a direct effect of IL-9 on ICACC-1 expression in the
lung, where IL-9 responsive cells contained within the lung express
ICACC-1.
Example 4
ICACC-1 Expression can be Induced in the Murine Lung by IL-9
[0158] ICACC-1 gene expression was assessed in vivo using the C57B6
mouse (bronchial hyporesponsive) which does not express detectable
levels of IL-9 and the DBA mouse (bronchial hyperresponsive) which
expresses robust levels of IL-9 (Nicolaides et al., 1997). RT-PCR
and Northern blot analysis of ICACC-1 from these lungs demonstrated
that ICACC-1 was expressed in the lung of mice which naturally
express high levels of IL-9 (DBA) but not in those with low levels
of IL-9 (C57B6) (FIG. 7).
[0159] To confirm that the expression of IL-9 was critically
related to the expression of ICACC-1 and to control for genetic
background specifically, recombinant murine IL-9 was introduced
into the lung of murine strain C57B6. Recombinant IL-9 was
instilled into the trachea of anesthetized mice by addition of 50
.mu.l of a 0.1 mg/ml IL 9 solution or vehicle alone (0.1% bovine
serum albumin) daily for ten days. After ten days, the mice were
euthanized and lungs extracted for either RNA expression analysis
using Trizol as described by the manufacturer (Gibco/BRL) or
Western blot analysis to determine levels of IL-9 instilled. The
Western blot analysis for IL-9 demonstrated that direct addition of
IL-9 to the lung resulted in an increase of overall amount of IL-9
in the lung while none was observed in the mouse instilled with
vehicle alone.
[0160] Expression of ICACC-1 RNA was measured as described in
Example 3. RT-PCR analysis for ICACC-1 RNA expression indicated
that expression increased when recombinant IL-9 was administered to
the lungs of the C57B6 mice, while no expression was observed in
the lungs of mice treated with vehicle only (FIG. 8). This data
demonstrates a direct role of IL-9 on inducing ICACC-1 expression
in the lung.
Example 5
Tissue Distribution of ICACC-1 in Mice
[0161] To address the possibility that ICACC-1 expression occurs
only in the presence of IL-9 expression, various organs were
extracted from Tg5 mice and analyzed for RNA expression via
Northern blot. BALBc mice were used as a control because they
express low levels of IL-9 in the lung when compared to Tg5 mice.
Tissue blots derived from BALBc murine organs were commercially
obtained (Clonetech) while tissue blots for Tg5 murine organs were
prepared by extracting organs followed by freezing in liquid
nitrogen. Total RNA was extracted from each of these organs using
Trizol as described by the manufacturer (Gibco/BRL). RNA was gel
electrophoresed and analyzed as described in Example 4. Lanes were
standardized by probing with .beta.-actin as an internal
control.
[0162] Tissue blots were probed using a DNA fragment comprising the
ICACC-1 cDNA. As shown in FIG. 9 (bottom), no signal was observed
in any of the tissues present on blots from normal mice. Analysis
of ICACC-1 expression in Tg5 organs revealed expression in the
lung, lymph node, colon, spleen, stomach, ovary and uterus (FIG. 9,
top). This data demonstrated that ICACC-1 is expressed in several
tissues in mice overexpressing IL-9 but not in those with low IL-9
levels. This data suggests that ICACC-1 may play a role in the
physiology of these organs in response to IL-9.
Example 5A
Induction of ICACC-1 in the Lung by Exposure to Antigen
[0163] Antigen sensitzation and phenotyping of animals was carried
out as previously described (McLane, M P, et al. Am. J. Respir.
Cell Mol. Biol. 19:713-720, 1998). Briefly, Balb/C mice were
intranasally exposed to Aspergillus fumagatus for 3-4 weeks. One
day after the final exposure, antigen exposed mice and naive
controls were phenotyped for bronchial hyperresponsiveness (BHR)
and cellularity in the airway. After phenotyping, organs were
removed and total RNA was prepared as described in Example 5 and
ICACC-1 expression was assessed in nave and antigen treated
tissues. As shown in FIG. 10, antigen exposed Balb/C mice had a
significant increase in BHR (FIG. 10A) and inflammatory cell influx
(the majority being eosinophils) as compared to controls (FIG.
10B). These features are very similar to clinical human asthma, and
reinforce the notion that this is a relevant model to study
molecular mechanisms and pharmaceutical target discovery for the
development of asthma drugs. ICACC-1 gene expression was tightly
associated with the asthmatic-like lung where robust expression was
found in the antigen treated lung (bottom panel, FIG. 11), while no
expression was found in the nave "normal" lung (top panel, FIG.
11). These data suggest that: 1) ICACC-1 is a potential therapeutic
target for the treatment of asthma, and 2) inhibiting the
expression or function of ICACC-1 will result in no toxic effects
to the lung.
Example 5B
Inhibition of Antigen Induced Induction of ICACC-1 in the Lung with
Anti-IL-9
[0164] IL-9 is a major mediator of the asthmatic response in man
and mouse models of asthma (Nicolaides, et al. Proc. Natl. Acad.
Sci. 94:13175-13180, 1997; McLane, M P, et al Am. J. Respir. Cell
Mol. Biol. 19:713-720, 1998; Temann et al., J. Exp. Med.
188:1307-1320, 1998: Levitt and Nicolaides, Emerg. Thera Targets
3:1-11, 1999). The use of IL-9 blocking antibodies in antigen
exposed mice suppresses the asthmatic-like phenotype (bronchial
hyperresponsiveness and influx of inflammatory cells such as
eosinophils). (B6D2)F1 mice were exposed to Aspergillus fumagatus
antigen as described in Example 5A on day 0, 7, 14, 21, and 22. A
subset of mice were also treated with 200 .mu.gs of anti-mIL9
(Pharmingen hamster antimouse IL-9) intra nasally on day 0, 7, 14,
and 21; or an isotype control Ig; or saline alone. All mice and
naive controls were phenotyped for BHR and BAL analysis as
described in Example 5A. As shown in FIG. 12A, anti-IL9 treatment
(Asp+.alpha.-mIL9) was able to significantly suppress BHR to levels
near that of naive, while isotype control Ig (Asp+Ig) had no effect
on reducing BHR. A similar result was found for airway eosinophilia
where a significant eosinophilia resulted upon antigen treatment
(Asp-) that was suppressed by anti-mIL9 treatment
(Asp+.alpha.-mIL9). Northern blot analysis of whole lungs from
these mice showed that anti-mIL9 also suppressed ICACC-1 gene
expression found in lungs of antigen exposed mice (FIG. 13). GADPH
which is a ubiquitously expressed house keeping gene was used as a
control to assure equal loading of RNA and overall gene expression.
Together, these data demonstrate a tight correlation of ICACC-1
gene expression and the asthmatic response. These data suggest that
blocking ICACC-1 expression or function would suppress the
asthmatic response.
Example 6
ICACC-1 Inducibility by IL-9 in Human Lung Epithelial Cells
[0165] To assess the ability of ICACC-1 to be induced by IL-9 in
epithelial cells, the human primary lung epithelial cell line NHBE
was assayed for expression levels of ICACC-1 in the presence of
IL-9. 1.times.10.sup.7 cells were harvested and washed three times
with phosphate-buffered saline and plated in medium in the presence
or absence of 50 ng/ml IL-9 for 72 hours. Cells were then harvested
and total RNA was extracted using Trizol as described by the
manufacturer (Gibco/BRL). RNA was processed and reverse transcribed
into cDNA as described in Example 3. Primers used to generate human
ICACC-1 message were; sense 5'-GATTCCAGGAACAGCTAAGC-3' (SEQ ID
NO:9) and antisense 5'-TATTTCATAGCTTGTAGCCTGG-3' (SEQ ID NO:10)
which produce a gene product of 722 bp. .gamma.-actin was assayed
as an internal control to measure for cDNA integrity using 10
primers previously described (Nicolaides et al., 1991). RT-PCR data
derived from human lung epithelial cells, shows that ICACC-1 is
induced in cells treated with IL-9 while no expression was observed
in untreated cells, indicating that the cells expressing ICACC-1
directly respond to IL-9 (FIG. 14).
[0166] Furthermore, human primary lung cultures that were
established from human lung biopsies were analyzed for IL-9 induced
expression of ICACC-1. Lung tissues were first minced with scissors
and passed through a wire mesh. Tissues were then digested with 175
iU/ml of collagenase (Sigma) for 1 hour at 37.degree. C. Tissue was
passed through 45 .mu.m and 15 .mu.m filters and then resuspended
in Dubelco Iscove's medium, and plated into 10 cm tissue culture
plates. Plates were incubated for 1 hour at 37.degree. C. to allow
macrophages to adhere to the plate and then non-adherent cells were
harvested and resuspended at 2.times.10.sup.5 cell/ml in Dulbelco
Iscoive's medium supplemented with 10% FBS, antibiotics and
cultured at 37.degree. C. with 5% CO.sub.2 for 4-5 days. For
ICACC-1 IL-9 induction studies, cells were incubated for 4-5 days
with or without 20 ng/ml recombinant human IL-9. Cells were then
harvested and total RNA was extracted by trizol as described above.
RNA was reverse transcribed and PCR'd for ICACC-1 using 5' primer
5'-CCCAAAGGAAGCCAACTCTGA-3" and 3' primer
5'-GTGAATGCCAGGAATGGTGCT-3' which resulted in a 253 bp product.
PMS2 which is a ubiquitously expressed house keeping gene was used
as an internal control as described (Nicolaides et al. Genomics 29:
329-334, 1995). Products were electrophoresed on 2% agarose gels
and visualized by ethidium bromide staining. As shown in FIG. 15,
IL-9 induced ICACC-1 expression in human primary lung cultures,
while cultures grown in the absence of IL-9 had no detectable
amounts of ICACC-1.
Example 6A
ICACC-1 Antiserum
[0167] Antisera were prepared to mICACC-1 by immunizing rabbits
with peptides selected from the mICACC-1 sequence using methods
described in Current Protocols in Immunology, Chapter 9, John Wiley
& Sons, Inc. The peptides used for the immunizations were;
residues 309-330, CLVLDKSGSMLNDDRLNRMNQA (SEQ ID NO: 11), residues
357-375, QSELKQLNSGADRDLLIKHC (SEQ ID NO: 12), residues 398422,
KKKYPTDGSEIVLLTDGEDNTISSC (SEQ ID NO: 13), residues 524-546,
TTHPPTIFIWDPSGVEQNGFILDC (SEQ ID NO: 14), residues 590-610,
CPPITVTPVVNKNTGKFPSPVT (SEQ ID NO: 15). The peptides were
synthesized by standard techniques of automated peptide synthesis
as either octavalent multiple antigen peptides (MAP) or as single
peptides. The single peptides were coupled to KLH for immunization
while the MAPs were used uncoupled. Rabbits were immunized with a
mixture of all five peptides either as KLH conjugates or MAPs. Both
immunogens produced useful antisera as indicated by their ability
to immunoprecipitate mICACC-1.
[0168] Immunoprecipitation of in vitro translated ICACC-1 was
performed to analyze the activities of these antibodies. A .sup.35S
labeled ICACC-1 fragment (429 amino acids in length corresponding
to amino acids 289-618 of the full length ICACC-1) was in vitro
translated using TNT Coupled Reticulocyte Lysate Systems (Promega).
Radio labeled ICACC-1 could be immunoprecipitated by 5 .mu.l of
ICACC-1 antisera or 1 .mu.g of protein A purified polyclonal
antibody. To assess the specificity of the ICACC-1 antisera, the
.sup.35S-labeled mIL-9 receptor fragment (60 KD mIL-9R) was used as
a negative control. Under the same precipitation conditions, none
of the mIL-9R protein was precipitated by the ICACC-1 antisera
(FIG. 16). These results indicated that antisera and protein A
purified polyclonal antibodies raised against ICACC-1 could
recognize ICACC-1 and therefore could potentially be used as
pharmaceutical reagents to block ICACC-1 function.
Example 6B
Suppression of IL-9 Induced Eotaxin Expression in the Lung using
Chloride Channel Blockers
[0169] IL-9 is known to induce eotaxin from lung epithelial cells
(Dong, et. al., submitted for publication, Eur. J. Immunol.). In
situ expression analysis of IL-9 transgenic mice found ICACC-1
expression to be predominant in airway epithelial cells. These
epithelial cells also produce eotaxin, and eotaxin can be induced
by IL-9 in these cells as well as primary lung cultures from a
variety of different mouse strains. Because eotaxin and ICACC-1 are
both induced by IL-9 in lung epithelial cells, it is possible that
inhibiting ICACC-1 can inhibit eotaxin or other cytokines such as
IL-4 or IL-13 (Doucet et al., J. Clin. Invest 101:2129-2139, 1998.)
that induce eotaxin production in lung epithelial cells. To test
this hypothesis, we employed a murine primary lung assays, where
lung cells were harvested from FVB/NJ mice as described in Example
SA and processed for in vitro analysis as described in Example 6
for human primary lung culture. Cells were incubated with or
without 20 ng/ml recombinant mIL-9 for 48 hours. After 48 hours,
conditioned supernatant was collected and analyzed for murine
eotaxin production using an eotaxin ELISA kit (R&D Systems).
Recombinant murine eotaxin was used to generate a standard curve.
As shown in FIG. 17, FVB primary cells when cultured with IL-9
produce up to 2 ng/ml of eotaxin in contrast to nearly undetectable
levels in FVB cultures grown in medium alone. A culture derived
from IL-9 transgenic mouse lung (TG5 lane) was used as a positive
control. This assay was used to assess the ability to suppress IL-9
induced eotaxin using chloride channel inhibitors DIDS and SITS.
Cultures were plated with or without mIL-9 in the presence of 0, 30
.mu.M and 100 .mu.M channel blocker. As shown in FIG. 18, eotaxin
production was inhibited 33% and 41% by 100 .mu.M DIDS or SITS
respectively. These data demonstrate the ability to suppress the
biological function of IL-9 on epithelial cells by inhibiting
chloride channel function. These data further indicate that
suppression of a chloride channel such as the asthma associated
ICACC-1 can result in a therapeutic benefit by the suppression of
antigen induced asthmatic responses. This screening assay and
technique can be used to evaluate other IL-9 induced genes whose
products are secreted proteins and is not restricted to using
eotaxin as the only marker. A similar approach will be taken using
the human ICACC-1 and human functional assays to identify
"specific" chloride channel inhibitors that suppress: 1) IL-9
induced effects such as de novo gene expression, and 2) ICACC-1
biologic function(s).
Example 7
Specific Blocking of ICACC-1 Signaling in vivo by Small Molecule
Inhibitors.
[0170] To demonstrate the specificity of ICACC-1 signaling which is
induced by IL-9, transfected cells expressing constitutively active
ICACC-1 are treated with chloride channel blocking compounds to
determine if inhibition of ICACC-1 blocks chloride channel
activity. Cells transfected with a constitutively activated ICACC-1
gene are plated at 3000 cells/well in the presence or absence of
IL-9 plus blocking compound and assessed for chloride channel
activity using a fluorescent chloride probe. Wild-type cells do not
exhibit the same amount of chloride channel activity as those
constitutively expressing ICACC-1. The addition of the blocking
compound on chloride channel activity is compared between wild-type
cells and those expressing constitutively activated ICACC-1.
Example 8
Blocking of ICACC-1 Induction by Aminosterols in Murine Lung
[0171] Lungs from the DBA bronchial hyperresponsive mouse are
treated with aminosterol compounds to test for their ability to
block expression of ICACC-1. This group of aminosterols was
identified from the liver of the dogfish shark as a class of
molecules that appear to be antiproliferative. An example of these
compounds are referred to in related U.S. patent application Ser.
No. 08/290,826. This series of aminosterols are assayed for their
ability to inhibit ICACC-1 expression and TH2 activity from the DBA
mouse as described below.
[0172] DBA mice are injected daily intraperitoneally with various
aminosterols at 10 mg/kg for 15 days. At day 15, mice are
phenotyped (see Example 9), euthanized and lungs extracted as
described in Example 1. RNA is isolated and processed for Northern
blot analysis using a ICACC-1 cDNA probe. The level of ICACC-1 RNA
detected by the probe indicates the extent of inhibition by
aminosterols when compared to control. The ability of specific
aminosterols, such as 1459, 1409,1436 and 1569, to block the
expression of ICACC-1 in vivo is assessed.
Example 9
Role of ICACC-1 in Murine Models of Asthma: Airway Response of
Unsensitized Animals
[0173] Certified virus-free male and female mice of the following
strains, DBA, C57B6 and B6D2F1 are purchased from the National
Cancer Institute or Jackson Laboratories (Bar Harbor Me.). IL-9
transgenic mice (Tg5) and their parent strain (FVB), are obtained
from the Ludwig Insitute (Brussels, Belgium). Animals are housed in
high-efficiency particulate filtered air laminar flow hoods in a
virus and antigen free facility and allowed free access to food and
water for 3 to 7 days prior to experimental manipulation. The
animal facilities are maintained at 22.degree. C. and the
light:dark cycle is automatically controlled (10:14 hour
light:dark).
[0174] Phenotyping and efficacy of pretreatment. To determine the
bronchoconstrictor response, respiratory system pressure is
measured at the trachea and recorded before and during exposure to
the drug. Mice are anesthetized and instrumented as previously
described. (Levitt et al., 1988; Levitt et al., 1989; Kleeberger et
al., 1990; Levitt et al., 1991; Levitt et al., 1995; Ewart et al,
1995). Airway responsiveness is measured to one or more of the
following: 5-hydroxytryptamine, acetylcholine, atracurium or a
substance-P analog. A simple and repeatable measure of the change
in peak inspiratory pressure following bronchoconstrictor challenge
is used which has been termed the Airway Pressure Time Index (APTI)
(Levitt et al., 1988; Levitt et al., 1989). The APTI is assessed by
the change in peak respiratory pressure integrated from the time of
injection until the peak pressure returns to baseline or plateau.
The APTI is comparable to airway resistance, however, the APTI
includes an additional component related to the recovery from
bronchoconstriction.
[0175] Prior to sacrifice, whole blood is collected for serum IgE
measurements by needle puncture of the inferior vena cava in
anesthetized animals. Samples are centrifuged to separate cells and
serum is collected and used to measure total IgE levels. Samples
not measured immediately are frozen at -20.degree. C.
[0176] All IgE serum samples are measured using an ELISA
antibody-sandwich assay. Microtiter plates are coated, 50 .mu.l per
well, with rat anti-murine IgE antibody (Southern Biotechnology) at
a concentration of 2.5 .mu.g/ml in a coating buffer of sodium
carbonate-sodium bicarbonate with sodium azide. Plates are covered
with plastic wrap and incubated at 4.degree. C. for 16 hours. The
plates are washed three times with a wash buffer of 0.05% Tween-20
in phosphate-buffered saline, incubating for five minutes for each
wash. Blocking of nonspecific binding sites is accomplished by
adding 200 .mu.l per well 5% bovine serum albumin in
phosphate-buffered saline, covering with plastic wrap and
incubating for 2 hours at 37.degree. C. After washing three times
with wash buffer, duplicate 50 .mu.l test samples are added to each
well. Test samples are assayed after being diluted 1:10, 1:50 and
1:100 with 5% bovine serun albumin in wash buffer. In addition to
the test samples, a set of IgE standards (PharMingen) at
concentrations from 0.8 ng/ml to 200 ng/ml in 5% bovine serum
albumin in wash buffer, are assayed to generate a standard curve. A
blank of no sample or standard is used to zero the plate reader
(background). After adding samples and standards, the plate is
covered with plastic wrap and incubated for 2 hours at room
temperature. After washing three times with wash buffer, 50 .mu.l
of secondary antibody rat anti-murine IgE-horseradish peroxidase
conjugate is added at a concentration of 250 ng/ml in 5% bovine
serum albumin in wash buffer. The plate is covered with plastic
wrap and incubated 2 hours at room temperature. After washing three
times with wash buffer, 100 .mu.l of the substrate 0.5 mg/ml
o-phenylenediamine in 0.1 M citrate buffer is added to every well.
After 5-10 minutes the reaction is stopped with 50 .mu.l of 12.5%
sulfuric acid and absorbance is measured at 490 nm on a MR5000
plate reader (Dynatech). A standard curve is constructed from the
standard IgE concentrations with antigen concentration on the x
axis (log scale) and absorbance on the y axis (linear scale). The
concentration of IgE in the samples is interpolated from the
standard curve.
[0177] Bronchoalveolar lavage and cellular analysis are preformed
as previously described (Kleeberger et al., 1990). Lung histology
is carried out after the lungs are extracted. Since prior
instrumentation may introduce artifact, separate animals are used
for these studies. Thus, a small group of animals is treated in
parallel exactly the same as the cohort undergoing various
pretreatments except these animals are not used for other tests
aside from bronchial responsiveness testing. After bronchial
responsiveness testing, the lungs are removed and submersed in
liquid nitrogen. Cryosectioning and histologic examination is
carried out in a manner obvious to those skilled in the art.
[0178] Polyclonal antibodies which block the murine ICACC-1 pathway
are used therapeutically to down-regulate the functions of, and
assess the importance of this pathway to bronchial responsiveness,
serum IgE and bronchoalveolar lavage in sensitized and unsensitized
mice. After antibody pretreatment, baseline bronchial
hyperresponsiveness, bronchoalveolar lavage and serum IgE levels
relative to Ig matched controls are determined.
Example 10
Role of ICACC-1 in Murine Models of Asthma: Airway Response of
Sensitized Animals
[0179] The data of Example 6a demonstrate that antisera is able to
be generated against ICACC-1 that recognizes the native protein
structure as determined by the ability to recognize the protein in
immunoprecipitation studies (FIG. 16). ICACC-1 blocking antibodies
represent potential therapeutic agents in suppressing the function
of ICACC-1. Studies are carried out using antigen sensitized
animals and protocols as described Examples 5A, 5B, and 10. Animals
are given ICACC-1 blocking antibodies via intranasal administration
as described in example SB and at day 23 animals are phenotyped for
BHR, BAL, and immunoglobulin levels. The effect of pretreatment
with ICACC-1 antibodies is used to assess the effect of
down-regulating ICACC-1 on the asthma phenotype.
[0180] While the invention has been described and illustrated
herein by references to various specific materials, procedures and
examples, it is understood that the invention is not restricted to
the particular combinations of material and procedures selected for
that purpose. Numerous variations of such details can be implied as
will be appreciated by those skilled in the art.
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Sequence CWU 1
1
18 1 2931 DNA Mus musculus CDS (8)..(2746) 1 ctgcagg atg gaa tct
ttg aag agt cct gtc ttc ctc ttg atc ctc cac 49 Met Glu Ser Leu Lys
Ser Pro Val Phe Leu Leu Ile Leu His 1 5 10 ctt ctg gaa gga gtt ctg
agt gag tcc ctc atc caa ctg aac aac aac 97 Leu Leu Glu Gly Val Leu
Ser Glu Ser Leu Ile Gln Leu Asn Asn Asn 15 20 25 30 ggc tat gag ggc
atc gtc atc gcc ata gac cac gac gtg ccg gaa gat 145 Gly Tyr Glu Gly
Ile Val Ile Ala Ile Asp His Asp Val Pro Glu Asp 35 40 45 gaa gcc
ctc att caa cac ata aag gac atg gtg act cag gcc tct cca 193 Glu Ala
Leu Ile Gln His Ile Lys Asp Met Val Thr Gln Ala Ser Pro 50 55 60
tac ctg ttt gaa gct aca gga aaa aga ttt tac ttc aaa aat gtt gcc 241
Tyr Leu Phe Glu Ala Thr Gly Lys Arg Phe Tyr Phe Lys Asn Val Ala 65
70 75 att ttg att ccc gag agc tgg aag gca aag cct gaa tat acg agg
cca 289 Ile Leu Ile Pro Glu Ser Trp Lys Ala Lys Pro Glu Tyr Thr Arg
Pro 80 85 90 aaa ctt gaa acc ttc aaa aac gct gat gtc ctt gta tca
aca acc agc 337 Lys Leu Glu Thr Phe Lys Asn Ala Asp Val Leu Val Ser
Thr Thr Ser 95 100 105 110 cct cta ggc aat gat gag ccc tac acc gaa
cat ata gga gca tgt gga 385 Pro Leu Gly Asn Asp Glu Pro Tyr Thr Glu
His Ile Gly Ala Cys Gly 115 120 125 gaa aag ggg atc agg att cac ctg
act cct gac ttc tta gca gga aag 433 Glu Lys Gly Ile Arg Ile His Leu
Thr Pro Asp Phe Leu Ala Gly Lys 130 135 140 aag ctg act cag tat ggg
cca caa gac agg acc ttt gtc cat gag tgg 481 Lys Leu Thr Gln Tyr Gly
Pro Gln Asp Arg Thr Phe Val His Glu Trp 145 150 155 gct cac ttc cga
tgg gga gtg ttt aat gaa tac aac aac gac gag aag 529 Ala His Phe Arg
Trp Gly Val Phe Asn Glu Tyr Asn Asn Asp Glu Lys 160 165 170 ttc tac
tta tcc aaa gga aaa ccc caa gca gtg agg tgt tca gca gcc 577 Phe Tyr
Leu Ser Lys Gly Lys Pro Gln Ala Val Arg Cys Ser Ala Ala 175 180 185
190 att acc ggt aaa aat caa gtt cgt cgg tgc cag gga ggc agt tgt atc
625 Ile Thr Gly Lys Asn Gln Val Arg Arg Cys Gln Gly Gly Ser Cys Ile
195 200 205 act aac gga aag tgt gta atc gac aga gta acg gga ctg tat
aaa gac 673 Thr Asn Gly Lys Cys Val Ile Asp Arg Val Thr Gly Leu Tyr
Lys Asp 210 215 220 aat tgt gta ttt gta cca gat cca cac caa aac gag
aag gct tcc atc 721 Asn Cys Val Phe Val Pro Asp Pro His Gln Asn Glu
Lys Ala Ser Ile 225 230 235 atg ttt aac caa aat atc aat tct gtg gtt
gaa ttc tgt aca gaa aaa 769 Met Phe Asn Gln Asn Ile Asn Ser Val Val
Glu Phe Cys Thr Glu Lys 240 245 250 aat cac aat caa gaa gcc cca aat
gac caa aac caa cga tgc aat ctc 817 Asn His Asn Gln Glu Ala Pro Asn
Asp Gln Asn Gln Arg Cys Asn Leu 255 260 265 270 cga agc acg tgg gaa
gtc atc cag gaa tct gag gac ttc aag caa acc 865 Arg Ser Thr Trp Glu
Val Ile Gln Glu Ser Glu Asp Phe Lys Gln Thr 275 280 285 act ccc atg
aca gcc cag cca cct gca ccc acc ttc tca ctg ctg caa 913 Thr Pro Met
Thr Ala Gln Pro Pro Ala Pro Thr Phe Ser Leu Leu Gln 290 295 300 att
gga caa aga att gtg tgc tta gtt ctt gat aag tcc ggg agc atg 961 Ile
Gly Gln Arg Ile Val Cys Leu Val Leu Asp Lys Ser Gly Ser Met 305 310
315 ctg aac gat gat cgt ctt aac cga atg aat cag gca agc cgg ctt ttc
1009 Leu Asn Asp Asp Arg Leu Asn Arg Met Asn Gln Ala Ser Arg Leu
Phe 320 325 330 ctg ctg cag act gtg gag cag gga tcc tgg gtc ggg atg
gtg acc ttt 1057 Leu Leu Gln Thr Val Glu Gln Gly Ser Trp Val Gly
Met Val Thr Phe 335 340 345 350 gac agt gct gcc tat gta caa agc gaa
ctc aaa cag tta aac agt ggt 1105 Asp Ser Ala Ala Tyr Val Gln Ser
Glu Leu Lys Gln Leu Asn Ser Gly 355 360 365 gct gac aga gat ctg ctg
atc aag cac tta ccc aca gta tct gca gga 1153 Ala Asp Arg Asp Leu
Leu Ile Lys His Leu Pro Thr Val Ser Ala Gly 370 375 380 ggg aca tct
ata tgc tct ggc ctt cgg aca gca ttt aca gtg ata aag 1201 Gly Thr
Ser Ile Cys Ser Gly Leu Arg Thr Ala Phe Thr Val Ile Lys 385 390 395
aag aag tat cca act gat gga tct gaa att gtg ctg ctg acc gat ggg
1249 Lys Lys Tyr Pro Thr Asp Gly Ser Glu Ile Val Leu Leu Thr Asp
Gly 400 405 410 gag gac aac acc att agc agc tgc ttt gac ctg gtg aag
cag agc ggg 1297 Glu Asp Asn Thr Ile Ser Ser Cys Phe Asp Leu Val
Lys Gln Ser Gly 415 420 425 430 gcc atc atc cat aca gtg gcc ctg gga
ccg gct gcc gct aaa gag ctt 1345 Ala Ile Ile His Thr Val Ala Leu
Gly Pro Ala Ala Ala Lys Glu Leu 435 440 445 gag cag ctg tcc aaa atg
aca gga ggc ctg cag aca tac tct tcg gat 1393 Glu Gln Leu Ser Lys
Met Thr Gly Gly Leu Gln Thr Tyr Ser Ser Asp 450 455 460 cag gtt cag
aac aat ggt ctt gtt gat gct ttc gca gca ctc tcc tca 1441 Gln Val
Gln Asn Asn Gly Leu Val Asp Ala Phe Ala Ala Leu Ser Ser 465 470 475
gga aat gcg gcg atc gct cag cac tcc atc cag ctg gag agc agg gga
1489 Gly Asn Ala Ala Ile Ala Gln His Ser Ile Gln Leu Glu Ser Arg
Gly 480 485 490 gtt aat ctc cag aat aac caa tgg atg aat ggc tca gtg
atc gtg gac 1537 Val Asn Leu Gln Asn Asn Gln Trp Met Asn Gly Ser
Val Ile Val Asp 495 500 505 510 agc tcg gtg ggc aag gac acc ttg ttt
ctt atc acc tgg aca acg cat 1585 Ser Ser Val Gly Lys Asp Thr Leu
Phe Leu Ile Thr Trp Thr Thr His 515 520 525 cct cct aca ata ttt atc
tgg gat ccc agc gga gtg gaa caa aat ggt 1633 Pro Pro Thr Ile Phe
Ile Trp Asp Pro Ser Gly Val Glu Gln Asn Gly 530 535 540 ttt ata cta
gac aca acc act aag gtg gcc tac ctc caa gtc cca ggc 1681 Phe Ile
Leu Asp Thr Thr Thr Lys Val Ala Tyr Leu Gln Val Pro Gly 545 550 555
acg gct aag gtt ggc ttt tgg aaa tac agc att caa gcg agc tca cag
1729 Thr Ala Lys Val Gly Phe Trp Lys Tyr Ser Ile Gln Ala Ser Ser
Gln 560 565 570 act ctc acc ttg act gtc acc tcc cgt gca gca agt gct
aca ctg cct 1777 Thr Leu Thr Leu Thr Val Thr Ser Arg Ala Ala Ser
Ala Thr Leu Pro 575 580 585 590 cct att aca gtg acc ccg gta gtg aat
aag aac aca ggg aaa ttc ccc 1825 Pro Ile Thr Val Thr Pro Val Val
Asn Lys Asn Thr Gly Lys Phe Pro 595 600 605 agc cct gta aca gtg tat
gca agc att cgc caa gga gcc tcg cct att 1873 Ser Pro Val Thr Val
Tyr Ala Ser Ile Arg Gln Gly Ala Ser Pro Ile 610 615 620 ctc agg gcc
agc gtc aca gcc ttg att gaa tct gtg aat gga aaa aca 1921 Leu Arg
Ala Ser Val Thr Ala Leu Ile Glu Ser Val Asn Gly Lys Thr 625 630 635
gta acc ctg gaa tta ctg gat aac gga gca ggt gcc gat gcc acc aag
1969 Val Thr Leu Glu Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Thr
Lys 640 645 650 aat gat ggt gtc tac tca agg ttt ttt aca gct ttt gat
gca aat ggt 2017 Asn Asp Gly Val Tyr Ser Arg Phe Phe Thr Ala Phe
Asp Ala Asn Gly 655 660 665 670 aga tac agc gtt aaa ata tgg gct ctg
gga gga gtc act tca gac aga 2065 Arg Tyr Ser Val Lys Ile Trp Ala
Leu Gly Gly Val Thr Ser Asp Arg 675 680 685 cag aga gca gca cct ccg
aag aac aga gcc atg tac ata gat ggc tgg 2113 Gln Arg Ala Ala Pro
Pro Lys Asn Arg Ala Met Tyr Ile Asp Gly Trp 690 695 700 att gag gat
ggt gaa gta aga atg aac cca cca cgt cct gaa act agt 2161 Ile Glu
Asp Gly Glu Val Arg Met Asn Pro Pro Arg Pro Glu Thr Ser 705 710 715
tat gtt caa gac aag cag ctg tgc ttc agc agg aca tct tca ggg gga
2209 Tyr Val Gln Asp Lys Gln Leu Cys Phe Ser Arg Thr Ser Ser Gly
Gly 720 725 730 tcg ttt gtg gcc acc aat gtc ccc gca gca gct ccc att
cct gac ctc 2257 Ser Phe Val Ala Thr Asn Val Pro Ala Ala Ala Pro
Ile Pro Asp Leu 735 740 745 750 ttt cca ccc tgt caa atc act gac ctg
aag gcc agc atc caa ggg cag 2305 Phe Pro Pro Cys Gln Ile Thr Asp
Leu Lys Ala Ser Ile Gln Gly Gln 755 760 765 aac ctg gtg aat ctg acg
tgg acg gct cct ggg gat gac tac gac cac 2353 Asn Leu Val Asn Leu
Thr Trp Thr Ala Pro Gly Asp Asp Tyr Asp His 770 775 780 ggg aga gct
tcc aac tac atc atc cga atg agc acc agt atc gtt gat 2401 Gly Arg
Ala Ser Asn Tyr Ile Ile Arg Met Ser Thr Ser Ile Val Asp 785 790 795
ctc agg gac cac ttc aac acc tca ctc caa gtg aac act acc ggt ctt
2449 Leu Arg Asp His Phe Asn Thr Ser Leu Gln Val Asn Thr Thr Gly
Leu 800 805 810 atc ccc aaa gag gcc agc tct gag gaa atc ttt gag ttt
gaa ctg gga 2497 Ile Pro Lys Glu Ala Ser Ser Glu Glu Ile Phe Glu
Phe Glu Leu Gly 815 820 825 830 ggc aac act ttt gga aat ggc aca gat
atc ttc att gct atc cag gct 2545 Gly Asn Thr Phe Gly Asn Gly Thr
Asp Ile Phe Ile Ala Ile Gln Ala 835 840 845 gtg gat aag tcc aat ctg
aaa tca gaa atc tcc aac att gca cgg gtg 2593 Val Asp Lys Ser Asn
Leu Lys Ser Glu Ile Ser Asn Ile Ala Arg Val 850 855 860 tct gtg ttc
atc ccc gct cag gag ccg ccc att ccc gaa gac tca act 2641 Ser Val
Phe Ile Pro Ala Gln Glu Pro Pro Ile Pro Glu Asp Ser Thr 865 870 875
ccc cct tgt cct gac atc agc atc aac agc acc att cct ggc atc cac
2689 Pro Pro Cys Pro Asp Ile Ser Ile Asn Ser Thr Ile Pro Gly Ile
His 880 885 890 gtg ctg aag ata atg tgg aag tgg cta ggg gaa atg cag
gtg aca cta 2737 Val Leu Lys Ile Met Trp Lys Trp Leu Gly Glu Met
Gln Val Thr Leu 895 900 905 910 ggt ttg cac tgaattttca ggcaagaaat
caaccagtca ttcctttcac 2786 Gly Leu His tggagaattt tctaaaaatg
tactttagac ttcctgtagg gggcggtata gtaacactcg 2846 aagctgtaaa
actgggtctg ggtgcattaa aaattatctg ttcaaataca aaaaaaaaaa 2906
aaaaaaaaaa aaaaaaaaaa aaaaa 2931 2 913 PRT Mus musculus 2 Met Glu
Ser Leu Lys Ser Pro Val Phe Leu Leu Ile Leu His Leu Leu 1 5 10 15
Glu Gly Val Leu Ser Glu Ser Leu Ile Gln Leu Asn Asn Asn Gly Tyr 20
25 30 Glu Gly Ile Val Ile Ala Ile Asp His Asp Val Pro Glu Asp Glu
Ala 35 40 45 Leu Ile Gln His Ile Lys Asp Met Val Thr Gln Ala Ser
Pro Tyr Leu 50 55 60 Phe Glu Ala Thr Gly Lys Arg Phe Tyr Phe Lys
Asn Val Ala Ile Leu 65 70 75 80 Ile Pro Glu Ser Trp Lys Ala Lys Pro
Glu Tyr Thr Arg Pro Lys Leu 85 90 95 Glu Thr Phe Lys Asn Ala Asp
Val Leu Val Ser Thr Thr Ser Pro Leu 100 105 110 Gly Asn Asp Glu Pro
Tyr Thr Glu His Ile Gly Ala Cys Gly Glu Lys 115 120 125 Gly Ile Arg
Ile His Leu Thr Pro Asp Phe Leu Ala Gly Lys Lys Leu 130 135 140 Thr
Gln Tyr Gly Pro Gln Asp Arg Thr Phe Val His Glu Trp Ala His 145 150
155 160 Phe Arg Trp Gly Val Phe Asn Glu Tyr Asn Asn Asp Glu Lys Phe
Tyr 165 170 175 Leu Ser Lys Gly Lys Pro Gln Ala Val Arg Cys Ser Ala
Ala Ile Thr 180 185 190 Gly Lys Asn Gln Val Arg Arg Cys Gln Gly Gly
Ser Cys Ile Thr Asn 195 200 205 Gly Lys Cys Val Ile Asp Arg Val Thr
Gly Leu Tyr Lys Asp Asn Cys 210 215 220 Val Phe Val Pro Asp Pro His
Gln Asn Glu Lys Ala Ser Ile Met Phe 225 230 235 240 Asn Gln Asn Ile
Asn Ser Val Val Glu Phe Cys Thr Glu Lys Asn His 245 250 255 Asn Gln
Glu Ala Pro Asn Asp Gln Asn Gln Arg Cys Asn Leu Arg Ser 260 265 270
Thr Trp Glu Val Ile Gln Glu Ser Glu Asp Phe Lys Gln Thr Thr Pro 275
280 285 Met Thr Ala Gln Pro Pro Ala Pro Thr Phe Ser Leu Leu Gln Ile
Gly 290 295 300 Gln Arg Ile Val Cys Leu Val Leu Asp Lys Ser Gly Ser
Met Leu Asn 305 310 315 320 Asp Asp Arg Leu Asn Arg Met Asn Gln Ala
Ser Arg Leu Phe Leu Leu 325 330 335 Gln Thr Val Glu Gln Gly Ser Trp
Val Gly Met Val Thr Phe Asp Ser 340 345 350 Ala Ala Tyr Val Gln Ser
Glu Leu Lys Gln Leu Asn Ser Gly Ala Asp 355 360 365 Arg Asp Leu Leu
Ile Lys His Leu Pro Thr Val Ser Ala Gly Gly Thr 370 375 380 Ser Ile
Cys Ser Gly Leu Arg Thr Ala Phe Thr Val Ile Lys Lys Lys 385 390 395
400 Tyr Pro Thr Asp Gly Ser Glu Ile Val Leu Leu Thr Asp Gly Glu Asp
405 410 415 Asn Thr Ile Ser Ser Cys Phe Asp Leu Val Lys Gln Ser Gly
Ala Ile 420 425 430 Ile His Thr Val Ala Leu Gly Pro Ala Ala Ala Lys
Glu Leu Glu Gln 435 440 445 Leu Ser Lys Met Thr Gly Gly Leu Gln Thr
Tyr Ser Ser Asp Gln Val 450 455 460 Gln Asn Asn Gly Leu Val Asp Ala
Phe Ala Ala Leu Ser Ser Gly Asn 465 470 475 480 Ala Ala Ile Ala Gln
His Ser Ile Gln Leu Glu Ser Arg Gly Val Asn 485 490 495 Leu Gln Asn
Asn Gln Trp Met Asn Gly Ser Val Ile Val Asp Ser Ser 500 505 510 Val
Gly Lys Asp Thr Leu Phe Leu Ile Thr Trp Thr Thr His Pro Pro 515 520
525 Thr Ile Phe Ile Trp Asp Pro Ser Gly Val Glu Gln Asn Gly Phe Ile
530 535 540 Leu Asp Thr Thr Thr Lys Val Ala Tyr Leu Gln Val Pro Gly
Thr Ala 545 550 555 560 Lys Val Gly Phe Trp Lys Tyr Ser Ile Gln Ala
Ser Ser Gln Thr Leu 565 570 575 Thr Leu Thr Val Thr Ser Arg Ala Ala
Ser Ala Thr Leu Pro Pro Ile 580 585 590 Thr Val Thr Pro Val Val Asn
Lys Asn Thr Gly Lys Phe Pro Ser Pro 595 600 605 Val Thr Val Tyr Ala
Ser Ile Arg Gln Gly Ala Ser Pro Ile Leu Arg 610 615 620 Ala Ser Val
Thr Ala Leu Ile Glu Ser Val Asn Gly Lys Thr Val Thr 625 630 635 640
Leu Glu Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Thr Lys Asn Asp 645
650 655 Gly Val Tyr Ser Arg Phe Phe Thr Ala Phe Asp Ala Asn Gly Arg
Tyr 660 665 670 Ser Val Lys Ile Trp Ala Leu Gly Gly Val Thr Ser Asp
Arg Gln Arg 675 680 685 Ala Ala Pro Pro Lys Asn Arg Ala Met Tyr Ile
Asp Gly Trp Ile Glu 690 695 700 Asp Gly Glu Val Arg Met Asn Pro Pro
Arg Pro Glu Thr Ser Tyr Val 705 710 715 720 Gln Asp Lys Gln Leu Cys
Phe Ser Arg Thr Ser Ser Gly Gly Ser Phe 725 730 735 Val Ala Thr Asn
Val Pro Ala Ala Ala Pro Ile Pro Asp Leu Phe Pro 740 745 750 Pro Cys
Gln Ile Thr Asp Leu Lys Ala Ser Ile Gln Gly Gln Asn Leu 755 760 765
Val Asn Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr Asp His Gly Arg 770
775 780 Ala Ser Asn Tyr Ile Ile Arg Met Ser Thr Ser Ile Val Asp Leu
Arg 785 790 795 800 Asp His Phe Asn Thr Ser Leu Gln Val Asn Thr Thr
Gly Leu Ile Pro 805 810 815 Lys Glu Ala Ser Ser Glu Glu Ile Phe Glu
Phe Glu Leu Gly Gly Asn 820 825 830 Thr Phe Gly Asn Gly Thr Asp Ile
Phe Ile Ala Ile Gln Ala Val Asp 835 840 845 Lys Ser Asn Leu Lys Ser
Glu Ile Ser Asn Ile Ala Arg Val Ser Val 850 855 860 Phe Ile Pro Ala
Gln Glu Pro Pro Ile Pro Glu Asp Ser Thr Pro Pro 865 870 875 880 Cys
Pro Asp Ile Ser Ile Asn Ser Thr Ile Pro Gly Ile His Val Leu 885 890
895 Lys Ile Met Trp Lys Trp Leu Gly Glu Met Gln Val Thr Leu Gly Leu
900 905 910 His 3 3190 DNA Homo sapiens CDS (120)..(2948) 3
cttcttgtgt tcttaaaccc ttgcaagttc agraagaaac ccatctgcat ccatattgaa
60
aacctgacac aatgtatgca gcaggctcag tgtgagtgaa ctggaggctt ctctacaac
119 atg acc caa agg agc att gca ggt cct att tgc aac ctg aag ttt gtg
167 Met Thr Gln Arg Ser Ile Ala Gly Pro Ile Cys Asn Leu Lys Phe Val
1 5 10 15 act ctc ctg gtt gcc tta agt tca gaa ctc cca ttc ctg gga
gct gga 215 Thr Leu Leu Val Ala Leu Ser Ser Glu Leu Pro Phe Leu Gly
Ala Gly 20 25 30 gta cag ctt caa gac aat ggg tat aat gga ttg ctc
att gca att aat 263 Val Gln Leu Gln Asp Asn Gly Tyr Asn Gly Leu Leu
Ile Ala Ile Asn 35 40 45 cct cag gta cct gag aat cag aac ctc atc
tca aac att aag gaa atg 311 Pro Gln Val Pro Glu Asn Gln Asn Leu Ile
Ser Asn Ile Lys Glu Met 50 55 60 ata act gaa gct tca ttt tac cta
ttt aat gct acc aag aga aga gta 359 Ile Thr Glu Ala Ser Phe Tyr Leu
Phe Asn Ala Thr Lys Arg Arg Val 65 70 75 80 ttt ttc aga aat ata aag
att tta ata cct gcc aca tgg aaa gct aat 407 Phe Phe Arg Asn Ile Lys
Ile Leu Ile Pro Ala Thr Trp Lys Ala Asn 85 90 95 aat aac agc aaa
ata aaa caa gaa tca tat gaa aag gca aat gtc ata 455 Asn Asn Ser Lys
Ile Lys Gln Glu Ser Tyr Glu Lys Ala Asn Val Ile 100 105 110 gtg act
gac tgg tat agg gca cat gga gat gat cca tac acc cta caa 503 Val Thr
Asp Trp Tyr Arg Ala His Gly Asp Asp Pro Tyr Thr Leu Gln 115 120 125
tac aga ggg tgt gga aaa gag gga aaa tac att cat ttc aca cct aat 551
Tyr Arg Gly Cys Gly Lys Glu Gly Lys Tyr Ile His Phe Thr Pro Asn 130
135 140 ttc cta ctg aat gat aac tta aca gct ggc tac gga tca cga ggc
cga 599 Phe Leu Leu Asn Asp Asn Leu Thr Ala Gly Tyr Gly Ser Arg Gly
Arg 145 150 155 160 gtg ttt gtc cat gaa tgg gcc cac ctc cgt tgg ggt
gtg ttc gat gag 647 Val Phe Val His Glu Trp Ala His Leu Arg Trp Gly
Val Phe Asp Glu 165 170 175 tat aac aat gac aaa cct ttc tac ata aat
ggg caa aat caa att aaa 695 Tyr Asn Asn Asp Lys Pro Phe Tyr Ile Asn
Gly Gln Asn Gln Ile Lys 180 185 190 gtg aca agg tgt tca tct gac atc
aca ggc att ttt gtg tgt gaa aaa 743 Val Thr Arg Cys Ser Ser Asp Ile
Thr Gly Ile Phe Val Cys Glu Lys 195 200 205 ggt cct tgc ccc caa gaa
aac tgt att att agt aag ctt ttt aaa gaa 791 Gly Pro Cys Pro Gln Glu
Asn Cys Ile Ile Ser Lys Leu Phe Lys Glu 210 215 220 gga tgc acc ttt
atc tac aat agc acc caa agt gca act gca tca ata 839 Gly Cys Thr Phe
Ile Tyr Asn Ser Thr Gln Ser Ala Thr Ala Ser Ile 225 230 235 240 atg
ttc atg cga agt tta tct tct gtg gtt gaa ttt tgt aat gca agt 887 Met
Phe Met Arg Ser Leu Ser Ser Val Val Glu Phe Cys Asn Ala Ser 245 250
255 acc cac aac caa gaa gca cca aac cta cag aac cag atg tgc agc ctc
935 Thr His Asn Gln Glu Ala Pro Asn Leu Gln Asn Gln Met Cys Ser Leu
260 265 270 aga agt gca tgg gat gta atc aca gac tct gct gac ttt cac
cac agc 983 Arg Ser Ala Trp Asp Val Ile Thr Asp Ser Ala Asp Phe His
His Ser 275 280 285 ttt ccc atg aac ggg act gag ctt cca cct cct ccc
aca ttc tcg ctt 1031 Phe Pro Met Asn Gly Thr Glu Leu Pro Pro Pro
Pro Thr Phe Ser Leu 290 295 300 gta gag gct ggt gac aaa gtg gtc tgt
tta gtg ctg gat gcg tcc agc 1079 Val Glu Ala Gly Asp Lys Val Val
Cys Leu Val Leu Asp Ala Ser Ser 305 310 315 320 aag atg gca gag gct
gac aga ctc ctt caa cta caa caa gcc gca gaa 1127 Lys Met Ala Glu
Ala Asp Arg Leu Leu Gln Leu Gln Gln Ala Ala Glu 325 330 335 ttt tat
ttg atg cag att gtt gaa att cat acc ttc gtg ggc att gcc 1175 Phe
Tyr Leu Met Gln Ile Val Glu Ile His Thr Phe Val Gly Ile Ala 340 345
350 agt ttc gac agc aaa gga gag atc aga gcc cag cta cac caa att aac
1223 Ser Phe Asp Ser Lys Gly Glu Ile Arg Ala Gln Leu His Gln Ile
Asn 355 360 365 agc aat gat gat cga aag ttg ctg gtt tca tat ctg ccc
acc act gta 1271 Ser Asn Asp Asp Arg Lys Leu Leu Val Ser Tyr Leu
Pro Thr Thr Val 370 375 380 tca gct aaa aca gac atc agc att tgt tca
ggg ctt aag aaa gga ttt 1319 Ser Ala Lys Thr Asp Ile Ser Ile Cys
Ser Gly Leu Lys Lys Gly Phe 385 390 395 400 gag gtg gtt gaa aaa ctg
aat gga aaa gct tat ggc tct gtg atg ata 1367 Glu Val Val Glu Lys
Leu Asn Gly Lys Ala Tyr Gly Ser Val Met Ile 405 410 415 tta gtg acc
agc gga gat gat aag ctt ctt ggc aat tgc tta ccc act 1415 Leu Val
Thr Ser Gly Asp Asp Lys Leu Leu Gly Asn Cys Leu Pro Thr 420 425 430
gtg ctc agc agt ggt tca aca att cac tcc att gcc ctg ggt tca tct
1463 Val Leu Ser Ser Gly Ser Thr Ile His Ser Ile Ala Leu Gly Ser
Ser 435 440 445 gca gcc cca aat ctg gag gaa tta tca cgt ctt aca gga
ggt tta aag 1511 Ala Ala Pro Asn Leu Glu Glu Leu Ser Arg Leu Thr
Gly Gly Leu Lys 450 455 460 ttc ttt gtt cca gat ata tca aac tcc aat
agc atg att gat gct ttc 1559 Phe Phe Val Pro Asp Ile Ser Asn Ser
Asn Ser Met Ile Asp Ala Phe 465 470 475 480 agt aga att tcc tct gga
act gga gac att ttc cag caa cat att cag 1607 Ser Arg Ile Ser Ser
Gly Thr Gly Asp Ile Phe Gln Gln His Ile Gln 485 490 495 ctt gaa agt
aca ggt gaa aat gtc aaa cct cac cat caa ttg aaa aac 1655 Leu Glu
Ser Thr Gly Glu Asn Val Lys Pro His His Gln Leu Lys Asn 500 505 510
aca gtg act gtg gat aat act gtg ggc aac gac act atg ttt cta gtt
1703 Thr Val Thr Val Asp Asn Thr Val Gly Asn Asp Thr Met Phe Leu
Val 515 520 525 acg tgg cag gcc agt ggt cct cct gag att ata tta ttt
gat cct gat 1751 Thr Trp Gln Ala Ser Gly Pro Pro Glu Ile Ile Leu
Phe Asp Pro Asp 530 535 540 gga cga aaa tac tac aca aat aat ttt atc
acc aat cta act ttt cgg 1799 Gly Arg Lys Tyr Tyr Thr Asn Asn Phe
Ile Thr Asn Leu Thr Phe Arg 545 550 555 560 aca gct agt ctt tgg att
cca gga aca gct aag cct ggg cac tgg act 1847 Thr Ala Ser Leu Trp
Ile Pro Gly Thr Ala Lys Pro Gly His Trp Thr 565 570 575 tac acc ctg
aac aat acc cat cat tct ctg caa gcc ctg aaa gtg aca 1895 Tyr Thr
Leu Asn Asn Thr His His Ser Leu Gln Ala Leu Lys Val Thr 580 585 590
gtg acc tct cgt gcc tcc aac tca gct gtg ccc cca gcc act gtg gaa
1943 Val Thr Ser Arg Ala Ser Asn Ser Ala Val Pro Pro Ala Thr Val
Glu 595 600 605 gcc ttt gtg gaa aga gac agc ctc cat ttt cct cat cct
gtg atg att 1991 Ala Phe Val Glu Arg Asp Ser Leu His Phe Pro His
Pro Val Met Ile 610 615 620 tat gcc aat gtg aaa cag gga ttt tat ccc
att ctt aat gcc act gtc 2039 Tyr Ala Asn Val Lys Gln Gly Phe Tyr
Pro Ile Leu Asn Ala Thr Val 625 630 635 640 act gcc aca gtt gag cca
gag act gga gat cct gtt acg ctg aga ctc 2087 Thr Ala Thr Val Glu
Pro Glu Thr Gly Asp Pro Val Thr Leu Arg Leu 645 650 655 ctt gat gat
gga gca ggt gct gat gtt ata aaa aat gat gga att tac 2135 Leu Asp
Asp Gly Ala Gly Ala Asp Val Ile Lys Asn Asp Gly Ile Tyr 660 665 670
tcg agg tat ttt ttc tcc ttt gct gca aat ggt aga tat agc ttg aaa
2183 Ser Arg Tyr Phe Phe Ser Phe Ala Ala Asn Gly Arg Tyr Ser Leu
Lys 675 680 685 gtg cat gtc aat cac tct ccc agc ata agc acc cca gcc
cac tct att 2231 Val His Val Asn His Ser Pro Ser Ile Ser Thr Pro
Ala His Ser Ile 690 695 700 cca ggg agt cat gct atg tat gta cca ggt
tac aca gca aac ggt aat 2279 Pro Gly Ser His Ala Met Tyr Val Pro
Gly Tyr Thr Ala Asn Gly Asn 705 710 715 720 att cag atg aat gct cca
agg aaa tca gta ggc aga aat gag gag gag 2327 Ile Gln Met Asn Ala
Pro Arg Lys Ser Val Gly Arg Asn Glu Glu Glu 725 730 735 cga aag tgg
ggc ttt agc cga gtc agc tca gga ggc tcc ttt tca gtg 2375 Arg Lys
Trp Gly Phe Ser Arg Val Ser Ser Gly Gly Ser Phe Ser Val 740 745 750
ctg gga gtt cca gct ggc ccc cac cct gat gtg ttt cca cca tgc aaa
2423 Leu Gly Val Pro Ala Gly Pro His Pro Asp Val Phe Pro Pro Cys
Lys 755 760 765 att att gac ctg gaa gct gta aaa gta gaa gag gaa ttg
acc cta tct 2471 Ile Ile Asp Leu Glu Ala Val Lys Val Glu Glu Glu
Leu Thr Leu Ser 770 775 780 tgg aca gca cct gga gaa gac ttt gat cag
ggc cag gct aca agc tat 2519 Trp Thr Ala Pro Gly Glu Asp Phe Asp
Gln Gly Gln Ala Thr Ser Tyr 785 790 795 800 gaa ata aga atg agt aaa
agt cta cag aat atc caa gat gac ttt aac 2567 Glu Ile Arg Met Ser
Lys Ser Leu Gln Asn Ile Gln Asp Asp Phe Asn 805 810 815 aat gct att
tta gta aat aca tca aag cga aat cct cag caa gct ggc 2615 Asn Ala
Ile Leu Val Asn Thr Ser Lys Arg Asn Pro Gln Gln Ala Gly 820 825 830
atc agg gag ata ttt acg ttc tca ccc cag att tcc acg aat gga cct
2663 Ile Arg Glu Ile Phe Thr Phe Ser Pro Gln Ile Ser Thr Asn Gly
Pro 835 840 845 gaa cat cag cca aat gga gaa aca cat gaa agc cac aga
att tat gtt 2711 Glu His Gln Pro Asn Gly Glu Thr His Glu Ser His
Arg Ile Tyr Val 850 855 860 gca ata cga gca atg gat agg aac tcc tta
cag tct gct gta tct aac 2759 Ala Ile Arg Ala Met Asp Arg Asn Ser
Leu Gln Ser Ala Val Ser Asn 865 870 875 880 att gcc cag gcg cct ctg
ttt att ccc ccc aat tct gat cct gta cct 2807 Ile Ala Gln Ala Pro
Leu Phe Ile Pro Pro Asn Ser Asp Pro Val Pro 885 890 895 gcc aga gat
tat ctt ata ttg aaa gga gtt tta aca gca atg ggt ttg 2855 Ala Arg
Asp Tyr Leu Ile Leu Lys Gly Val Leu Thr Ala Met Gly Leu 900 905 910
ata gga atc att tgc ctt att ata gtt gtg aca cat cat act tta agc
2903 Ile Gly Ile Ile Cys Leu Ile Ile Val Val Thr His His Thr Leu
Ser 915 920 925 agg aaa aag aga gca gac aag aaa gag aat gga aca aaa
tta tta 2948 Arg Lys Lys Arg Ala Asp Lys Lys Glu Asn Gly Thr Lys
Leu Leu 930 935 940 taaataaata tccaaagtgt cttccttctt agatataaga
cccatggcct tcgactacaa 3008 aaacatacta acaaagtcaa attaacatca
aaactgtatt aaaatgcatt gagttttgta 3068 caatacagat aagattttta
catggtagat caacaaattc tttttggggg tagattagaa 3128 aaccttacac
tttggctatg aacaaataat aaaaattatt ctttaaaaaa aaaaaaaaaa 3188 aa 3190
4 943 PRT Homo sapiens 4 Met Thr Gln Arg Ser Ile Ala Gly Pro Ile
Cys Asn Leu Lys Phe Val 1 5 10 15 Thr Leu Leu Val Ala Leu Ser Ser
Glu Leu Pro Phe Leu Gly Ala Gly 20 25 30 Val Gln Leu Gln Asp Asn
Gly Tyr Asn Gly Leu Leu Ile Ala Ile Asn 35 40 45 Pro Gln Val Pro
Glu Asn Gln Asn Leu Ile Ser Asn Ile Lys Glu Met 50 55 60 Ile Thr
Glu Ala Ser Phe Tyr Leu Phe Asn Ala Thr Lys Arg Arg Val 65 70 75 80
Phe Phe Arg Asn Ile Lys Ile Leu Ile Pro Ala Thr Trp Lys Ala Asn 85
90 95 Asn Asn Ser Lys Ile Lys Gln Glu Ser Tyr Glu Lys Ala Asn Val
Ile 100 105 110 Val Thr Asp Trp Tyr Arg Ala His Gly Asp Asp Pro Tyr
Thr Leu Gln 115 120 125 Tyr Arg Gly Cys Gly Lys Glu Gly Lys Tyr Ile
His Phe Thr Pro Asn 130 135 140 Phe Leu Leu Asn Asp Asn Leu Thr Ala
Gly Tyr Gly Ser Arg Gly Arg 145 150 155 160 Val Phe Val His Glu Trp
Ala His Leu Arg Trp Gly Val Phe Asp Glu 165 170 175 Tyr Asn Asn Asp
Lys Pro Phe Tyr Ile Asn Gly Gln Asn Gln Ile Lys 180 185 190 Val Thr
Arg Cys Ser Ser Asp Ile Thr Gly Ile Phe Val Cys Glu Lys 195 200 205
Gly Pro Cys Pro Gln Glu Asn Cys Ile Ile Ser Lys Leu Phe Lys Glu 210
215 220 Gly Cys Thr Phe Ile Tyr Asn Ser Thr Gln Ser Ala Thr Ala Ser
Ile 225 230 235 240 Met Phe Met Arg Ser Leu Ser Ser Val Val Glu Phe
Cys Asn Ala Ser 245 250 255 Thr His Asn Gln Glu Ala Pro Asn Leu Gln
Asn Gln Met Cys Ser Leu 260 265 270 Arg Ser Ala Trp Asp Val Ile Thr
Asp Ser Ala Asp Phe His His Ser 275 280 285 Phe Pro Met Asn Gly Thr
Glu Leu Pro Pro Pro Pro Thr Phe Ser Leu 290 295 300 Val Glu Ala Gly
Asp Lys Val Val Cys Leu Val Leu Asp Ala Ser Ser 305 310 315 320 Lys
Met Ala Glu Ala Asp Arg Leu Leu Gln Leu Gln Gln Ala Ala Glu 325 330
335 Phe Tyr Leu Met Gln Ile Val Glu Ile His Thr Phe Val Gly Ile Ala
340 345 350 Ser Phe Asp Ser Lys Gly Glu Ile Arg Ala Gln Leu His Gln
Ile Asn 355 360 365 Ser Asn Asp Asp Arg Lys Leu Leu Val Ser Tyr Leu
Pro Thr Thr Val 370 375 380 Ser Ala Lys Thr Asp Ile Ser Ile Cys Ser
Gly Leu Lys Lys Gly Phe 385 390 395 400 Glu Val Val Glu Lys Leu Asn
Gly Lys Ala Tyr Gly Ser Val Met Ile 405 410 415 Leu Val Thr Ser Gly
Asp Asp Lys Leu Leu Gly Asn Cys Leu Pro Thr 420 425 430 Val Leu Ser
Ser Gly Ser Thr Ile His Ser Ile Ala Leu Gly Ser Ser 435 440 445 Ala
Ala Pro Asn Leu Glu Glu Leu Ser Arg Leu Thr Gly Gly Leu Lys 450 455
460 Phe Phe Val Pro Asp Ile Ser Asn Ser Asn Ser Met Ile Asp Ala Phe
465 470 475 480 Ser Arg Ile Ser Ser Gly Thr Gly Asp Ile Phe Gln Gln
His Ile Gln 485 490 495 Leu Glu Ser Thr Gly Glu Asn Val Lys Pro His
His Gln Leu Lys Asn 500 505 510 Thr Val Thr Val Asp Asn Thr Val Gly
Asn Asp Thr Met Phe Leu Val 515 520 525 Thr Trp Gln Ala Ser Gly Pro
Pro Glu Ile Ile Leu Phe Asp Pro Asp 530 535 540 Gly Arg Lys Tyr Tyr
Thr Asn Asn Phe Ile Thr Asn Leu Thr Phe Arg 545 550 555 560 Thr Ala
Ser Leu Trp Ile Pro Gly Thr Ala Lys Pro Gly His Trp Thr 565 570 575
Tyr Thr Leu Asn Asn Thr His His Ser Leu Gln Ala Leu Lys Val Thr 580
585 590 Val Thr Ser Arg Ala Ser Asn Ser Ala Val Pro Pro Ala Thr Val
Glu 595 600 605 Ala Phe Val Glu Arg Asp Ser Leu His Phe Pro His Pro
Val Met Ile 610 615 620 Tyr Ala Asn Val Lys Gln Gly Phe Tyr Pro Ile
Leu Asn Ala Thr Val 625 630 635 640 Thr Ala Thr Val Glu Pro Glu Thr
Gly Asp Pro Val Thr Leu Arg Leu 645 650 655 Leu Asp Asp Gly Ala Gly
Ala Asp Val Ile Lys Asn Asp Gly Ile Tyr 660 665 670 Ser Arg Tyr Phe
Phe Ser Phe Ala Ala Asn Gly Arg Tyr Ser Leu Lys 675 680 685 Val His
Val Asn His Ser Pro Ser Ile Ser Thr Pro Ala His Ser Ile 690 695 700
Pro Gly Ser His Ala Met Tyr Val Pro Gly Tyr Thr Ala Asn Gly Asn 705
710 715 720 Ile Gln Met Asn Ala Pro Arg Lys Ser Val Gly Arg Asn Glu
Glu Glu 725 730 735 Arg Lys Trp Gly Phe Ser Arg Val Ser Ser Gly Gly
Ser Phe Ser Val 740 745 750 Leu Gly Val Pro Ala Gly Pro His Pro Asp
Val Phe Pro Pro Cys Lys 755 760 765 Ile Ile Asp Leu Glu Ala Val Lys
Val Glu Glu Glu Leu Thr Leu Ser 770 775 780 Trp Thr Ala Pro Gly Glu
Asp Phe Asp Gln Gly Gln Ala Thr Ser Tyr 785 790 795 800 Glu Ile Arg
Met Ser Lys Ser Leu Gln Asn Ile Gln Asp Asp Phe Asn 805 810 815 Asn
Ala Ile Leu Val Asn Thr Ser Lys Arg Asn Pro Gln Gln Ala Gly 820 825
830 Ile Arg Glu Ile Phe Thr Phe Ser Pro Gln Ile Ser Thr Asn Gly Pro
835 840 845 Glu His Gln Pro Asn Gly Glu Thr His Glu Ser His Arg Ile
Tyr Val 850 855 860 Ala Ile Arg Ala Met Asp Arg Asn Ser Leu Gln
Ser
Ala Val Ser Asn 865 870 875 880 Ile Ala Gln Ala Pro Leu Phe Ile Pro
Pro Asn Ser Asp Pro Val Pro 885 890 895 Ala Arg Asp Tyr Leu Ile Leu
Lys Gly Val Leu Thr Ala Met Gly Leu 900 905 910 Ile Gly Ile Ile Cys
Leu Ile Ile Val Val Thr His His Thr Leu Ser 915 920 925 Arg Lys Lys
Arg Ala Asp Lys Lys Glu Asn Gly Thr Lys Leu Leu 930 935 940 5 2745
DNA Homo sapiens CDS (1)..(2742) 5 atg ggg cca ttt aag agt tct gtg
ttc atc ttg att ctt cac ctt cta 48 Met Gly Pro Phe Lys Ser Ser Val
Phe Ile Leu Ile Leu His Leu Leu 1 5 10 15 gaa ggg gcc ctg agt aat
tca ctc att cag ctg aac aac aat ggc tat 96 Glu Gly Ala Leu Ser Asn
Ser Leu Ile Gln Leu Asn Asn Asn Gly Tyr 20 25 30 gaa ggc att gtc
gtt gca atc gac ccc aat gtg cca gaa gat gaa aca 144 Glu Gly Ile Val
Val Ala Ile Asp Pro Asn Val Pro Glu Asp Glu Thr 35 40 45 ctc att
caa caa ata aag gac atg gtg acc cag gca tct ctg tat ctg 192 Leu Ile
Gln Gln Ile Lys Asp Met Val Thr Gln Ala Ser Leu Tyr Leu 50 55 60
ttt gaa gct aca gga aag cga ttt tat ttc aaa aat gtt gcc att ttg 240
Phe Glu Ala Thr Gly Lys Arg Phe Tyr Phe Lys Asn Val Ala Ile Leu 65
70 75 80 att cct gaa aca tgg aag aca aag gct gac tat gtg aga cca
aaa ctt 288 Ile Pro Glu Thr Trp Lys Thr Lys Ala Asp Tyr Val Arg Pro
Lys Leu 85 90 95 gag acc tac aaa aat gct gat gtt ctg gtt gct gag
tct act cct cca 336 Glu Thr Tyr Lys Asn Ala Asp Val Leu Val Ala Glu
Ser Thr Pro Pro 100 105 110 ggt aat gat gaa ccc tac act gag cag atg
ggc aac tgt gga gag aag 384 Gly Asn Asp Glu Pro Tyr Thr Glu Gln Met
Gly Asn Cys Gly Glu Lys 115 120 125 ggt gaa agg atc cac ctc act cct
gat ttc att gca gga aaa aag tta 432 Gly Glu Arg Ile His Leu Thr Pro
Asp Phe Ile Ala Gly Lys Lys Leu 130 135 140 gct gaa tat gga cca caa
ggt agg gca ttt gtc cat gag tgg gct cat 480 Ala Glu Tyr Gly Pro Gln
Gly Arg Ala Phe Val His Glu Trp Ala His 145 150 155 160 cta cga tgg
gga gta ttt gac gag tac aat aat gat gag aaa ttc tac 528 Leu Arg Trp
Gly Val Phe Asp Glu Tyr Asn Asn Asp Glu Lys Phe Tyr 165 170 175 tta
tcc aat gga aga ata caa gca gta aga tgt tca gca ggt att act 576 Leu
Ser Asn Gly Arg Ile Gln Ala Val Arg Cys Ser Ala Gly Ile Thr 180 185
190 ggt aca aat gta gta aag aag tgt cag gga ggc agc tgt tac acc aaa
624 Gly Thr Asn Val Val Lys Lys Cys Gln Gly Gly Ser Cys Tyr Thr Lys
195 200 205 aga tgc aca ttc aat aaa gtw aca gga ctc tat gaa aaa gga
tgt gag 672 Arg Cys Thr Phe Asn Lys Xaa Thr Gly Leu Tyr Glu Lys Gly
Cys Glu 210 215 220 ttt gtt ctc caa tcc cgc cag acg gag aag gct tct
ata atg ttt gca 720 Phe Val Leu Gln Ser Arg Gln Thr Glu Lys Ala Ser
Ile Met Phe Ala 225 230 235 240 caa cat gtt gat tct ata gtt gaa ttc
tgt aca gaa caa aac cac aac 768 Gln His Val Asp Ser Ile Val Glu Phe
Cys Thr Glu Gln Asn His Asn 245 250 255 aaa gaa gct cca aac aag caa
aat caa aaa tgc aat ctc cga agc aca 816 Lys Glu Ala Pro Asn Lys Gln
Asn Gln Lys Cys Asn Leu Arg Ser Thr 260 265 270 tgg gaa gtg atc cgt
gat tct gag gac ttt aag aaa acc act cct atg 864 Trp Glu Val Ile Arg
Asp Ser Glu Asp Phe Lys Lys Thr Thr Pro Met 275 280 285 aca aca cag
cca cca aat ccc acc ttc tca ttg ctg cag att gga caa 912 Thr Thr Gln
Pro Pro Asn Pro Thr Phe Ser Leu Leu Gln Ile Gly Gln 290 295 300 aga
att gtg tgt tta gtc ctt gac aaa tct gga agc atg gcg act ggt 960 Arg
Ile Val Cys Leu Val Leu Asp Lys Ser Gly Ser Met Ala Thr Gly 305 310
315 320 aac cgc ctc aat cga ctg aat caa gca ggc cag ctt ttc ctg ctg
cag 1008 Asn Arg Leu Asn Arg Leu Asn Gln Ala Gly Gln Leu Phe Leu
Leu Gln 325 330 335 aca gtt gag ctg ggg tcc tgg gtt ggg atg gtg aca
ttt gac agt gct 1056 Thr Val Glu Leu Gly Ser Trp Val Gly Met Val
Thr Phe Asp Ser Ala 340 345 350 gcc cat gta caa agt gaa ctc ata cag
ata aac agt ggc agt gac agg 1104 Ala His Val Gln Ser Glu Leu Ile
Gln Ile Asn Ser Gly Ser Asp Arg 355 360 365 gac aca ctc gcc aaa aga
tta cct gca gca gct tca gga ggg acg tcc 1152 Asp Thr Leu Ala Lys
Arg Leu Pro Ala Ala Ala Ser Gly Gly Thr Ser 370 375 380 atc tgc agc
ggg ctt cga tcg gca ttt act gtg att agg aag aaa tat 1200 Ile Cys
Ser Gly Leu Arg Ser Ala Phe Thr Val Ile Arg Lys Lys Tyr 385 390 395
400 cca act gat gga tct gaa att gtg ctg ctg acg gat ggg gaa gac aac
1248 Pro Thr Asp Gly Ser Glu Ile Val Leu Leu Thr Asp Gly Glu Asp
Asn 405 410 415 act ata agt ggg tgc ttt aac gag gtc aaa caa agt ggt
gcc atc atc 1296 Thr Ile Ser Gly Cys Phe Asn Glu Val Lys Gln Ser
Gly Ala Ile Ile 420 425 430 cac aca gtc gct ttg ggg ccc tct gca gct
caa gaa cta gag gag ctg 1344 His Thr Val Ala Leu Gly Pro Ser Ala
Ala Gln Glu Leu Glu Glu Leu 435 440 445 tcc aaa atg aca gga ggt tta
cag aca tat gct tca gat caa gtt cag 1392 Ser Lys Met Thr Gly Gly
Leu Gln Thr Tyr Ala Ser Asp Gln Val Gln 450 455 460 aac aat ggc ctc
att gat gct ttt ggg gcc ctt tca tca gga aat gga 1440 Asn Asn Gly
Leu Ile Asp Ala Phe Gly Ala Leu Ser Ser Gly Asn Gly 465 470 475 480
gct gtc tct cag cgc tcc atc cag ctt gag agt aag gga tta acc ctc
1488 Ala Val Ser Gln Arg Ser Ile Gln Leu Glu Ser Lys Gly Leu Thr
Leu 485 490 495 cag aac agc cag tgg atg aat ggc aca gtg atc gtg gac
agc acc gtg 1536 Gln Asn Ser Gln Trp Met Asn Gly Thr Val Ile Val
Asp Ser Thr Val 500 505 510 gga aag gac act ttg ttt ctt atc acc tgg
aca acg cag cct ccc caa 1584 Gly Lys Asp Thr Leu Phe Leu Ile Thr
Trp Thr Thr Gln Pro Pro Gln 515 520 525 atc ctt ctc tgg gat ccc agt
gga cag aag caa ggt ggc ttt gta gtg 1632 Ile Leu Leu Trp Asp Pro
Ser Gly Gln Lys Gln Gly Gly Phe Val Val 530 535 540 gac aaa aac acc
aaa atg gcc tac ctc caa atc cca ggc att gct aag 1680 Asp Lys Asn
Thr Lys Met Ala Tyr Leu Gln Ile Pro Gly Ile Ala Lys 545 550 555 560
gtt ggc act tgg aaa tac agt ctg caa gca agc tca caa acc ttg acc
1728 Val Gly Thr Trp Lys Tyr Ser Leu Gln Ala Ser Ser Gln Thr Leu
Thr 565 570 575 ctg act gtc acg tcc cgt gcg tcc aat gct acc ctg cct
cca att aca 1776 Leu Thr Val Thr Ser Arg Ala Ser Asn Ala Thr Leu
Pro Pro Ile Thr 580 585 590 gtg act tcc aaa acg aac aag gac acc agc
aaa ttc ccc agc cct ctg 1824 Val Thr Ser Lys Thr Asn Lys Asp Thr
Ser Lys Phe Pro Ser Pro Leu 595 600 605 gta gtt tat gca aat att cgc
caa gga gcc tcc cca att ctc agg gcc 1872 Val Val Tyr Ala Asn Ile
Arg Gln Gly Ala Ser Pro Ile Leu Arg Ala 610 615 620 agt gtc aca gcc
ctg att gaa tca gtg aat gga aaa aca gtt acc ttg 1920 Ser Val Thr
Ala Leu Ile Glu Ser Val Asn Gly Lys Thr Val Thr Leu 625 630 635 640
gaa cta ctg gat aat gga gca ggt gct gat gct act aag gat gac ggt
1968 Glu Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Thr Lys Asp Asp
Gly 645 650 655 gtc tac tca agg tat ttc aca act tat gac acg aat ggt
aga tac agt 2016 Val Tyr Ser Arg Tyr Phe Thr Thr Tyr Asp Thr Asn
Gly Arg Tyr Ser 660 665 670 gta aaa gtg cgg gct ctg gga gga gtt aac
gca gcc aga cgg aga gtg 2064 Val Lys Val Arg Ala Leu Gly Gly Val
Asn Ala Ala Arg Arg Arg Val 675 680 685 ata ccc cag cag agt gga gca
ctg tac ata cct ggc tgg att gag aat 2112 Ile Pro Gln Gln Ser Gly
Ala Leu Tyr Ile Pro Gly Trp Ile Glu Asn 690 695 700 gat gaa atc caa
tgg aat cca cca aga cct gaa att aat aag gat gat 2160 Asp Glu Ile
Gln Trp Asn Pro Pro Arg Pro Glu Ile Asn Lys Asp Asp 705 710 715 720
gtt caa cac aag caa gtg tgt ttc agc aga aca tcc tcg gga ggc tca
2208 Val Gln His Lys Gln Val Cys Phe Ser Arg Thr Ser Ser Gly Gly
Ser 725 730 735 ttt gtg gct tct gat gtc cca aat gct ccc ata cct gat
ctc ttc cca 2256 Phe Val Ala Ser Asp Val Pro Asn Ala Pro Ile Pro
Asp Leu Phe Pro 740 745 750 cct ggc caa atc acc gac ctg aag gcg gaa
att cac ggg ggc agt ctc 2304 Pro Gly Gln Ile Thr Asp Leu Lys Ala
Glu Ile His Gly Gly Ser Leu 755 760 765 att aat ctg act tgg aca gct
cct ggg gat gat tat gac cat gga aca 2352 Ile Asn Leu Thr Trp Thr
Ala Pro Gly Asp Asp Tyr Asp His Gly Thr 770 775 780 gct cac aag tat
atc att cga ata agt aca agt att ctt gat ctc aga 2400 Ala His Lys
Tyr Ile Ile Arg Ile Ser Thr Ser Ile Leu Asp Leu Arg 785 790 795 800
gac aag ttc aat gaa tct ctt caa gtg aat act act gct ctc atc cca
2448 Asp Lys Phe Asn Glu Ser Leu Gln Val Asn Thr Thr Ala Leu Ile
Pro 805 810 815 aag gaa gcc aac tct gag gaa gtc ttt ttg ttt aaa cca
gaa aac att 2496 Lys Glu Ala Asn Ser Glu Glu Val Phe Leu Phe Lys
Pro Glu Asn Ile 820 825 830 act ttt gaa aat ggc aca gat ctt ttc att
gct att cag gct gtt gat 2544 Thr Phe Glu Asn Gly Thr Asp Leu Phe
Ile Ala Ile Gln Ala Val Asp 835 840 845 aag gtc gat ctg aaa tca gaa
ata tcc aac att gca cga gta tct ttg 2592 Lys Val Asp Leu Lys Ser
Glu Ile Ser Asn Ile Ala Arg Val Ser Leu 850 855 860 ttt att cct cca
cag act ccg cca gag aca cct agt cct gat gaa acg 2640 Phe Ile Pro
Pro Gln Thr Pro Pro Glu Thr Pro Ser Pro Asp Glu Thr 865 870 875 880
tct gct cct tgt cct aat att cat atc aac agc acc att cct ggc att
2688 Ser Ala Pro Cys Pro Asn Ile His Ile Asn Ser Thr Ile Pro Gly
Ile 885 890 895 cac att tta aaa att atg tgg aag tgg ata gga gaa ctg
cag ctg tca 2736 His Ile Leu Lys Ile Met Trp Lys Trp Ile Gly Glu
Leu Gln Leu Ser 900 905 910 ata gcc tag 2745 Ile Ala 6 914 PRT Homo
sapiens 6 Met Gly Pro Phe Lys Ser Ser Val Phe Ile Leu Ile Leu His
Leu Leu 1 5 10 15 Glu Gly Ala Leu Ser Asn Ser Leu Ile Gln Leu Asn
Asn Asn Gly Tyr 20 25 30 Glu Gly Ile Val Val Ala Ile Asp Pro Asn
Val Pro Glu Asp Glu Thr 35 40 45 Leu Ile Gln Gln Ile Lys Asp Met
Val Thr Gln Ala Ser Leu Tyr Leu 50 55 60 Phe Glu Ala Thr Gly Lys
Arg Phe Tyr Phe Lys Asn Val Ala Ile Leu 65 70 75 80 Ile Pro Glu Thr
Trp Lys Thr Lys Ala Asp Tyr Val Arg Pro Lys Leu 85 90 95 Glu Thr
Tyr Lys Asn Ala Asp Val Leu Val Ala Glu Ser Thr Pro Pro 100 105 110
Gly Asn Asp Glu Pro Tyr Thr Glu Gln Met Gly Asn Cys Gly Glu Lys 115
120 125 Gly Glu Arg Ile His Leu Thr Pro Asp Phe Ile Ala Gly Lys Lys
Leu 130 135 140 Ala Glu Tyr Gly Pro Gln Gly Arg Ala Phe Val His Glu
Trp Ala His 145 150 155 160 Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn
Asn Asp Glu Lys Phe Tyr 165 170 175 Leu Ser Asn Gly Arg Ile Gln Ala
Val Arg Cys Ser Ala Gly Ile Thr 180 185 190 Gly Thr Asn Val Val Lys
Lys Cys Gln Gly Gly Ser Cys Tyr Thr Lys 195 200 205 Arg Cys Thr Phe
Asn Lys Xaa Thr Gly Leu Tyr Glu Lys Gly Cys Glu 210 215 220 Phe Val
Leu Gln Ser Arg Gln Thr Glu Lys Ala Ser Ile Met Phe Ala 225 230 235
240 Gln His Val Asp Ser Ile Val Glu Phe Cys Thr Glu Gln Asn His Asn
245 250 255 Lys Glu Ala Pro Asn Lys Gln Asn Gln Lys Cys Asn Leu Arg
Ser Thr 260 265 270 Trp Glu Val Ile Arg Asp Ser Glu Asp Phe Lys Lys
Thr Thr Pro Met 275 280 285 Thr Thr Gln Pro Pro Asn Pro Thr Phe Ser
Leu Leu Gln Ile Gly Gln 290 295 300 Arg Ile Val Cys Leu Val Leu Asp
Lys Ser Gly Ser Met Ala Thr Gly 305 310 315 320 Asn Arg Leu Asn Arg
Leu Asn Gln Ala Gly Gln Leu Phe Leu Leu Gln 325 330 335 Thr Val Glu
Leu Gly Ser Trp Val Gly Met Val Thr Phe Asp Ser Ala 340 345 350 Ala
His Val Gln Ser Glu Leu Ile Gln Ile Asn Ser Gly Ser Asp Arg 355 360
365 Asp Thr Leu Ala Lys Arg Leu Pro Ala Ala Ala Ser Gly Gly Thr Ser
370 375 380 Ile Cys Ser Gly Leu Arg Ser Ala Phe Thr Val Ile Arg Lys
Lys Tyr 385 390 395 400 Pro Thr Asp Gly Ser Glu Ile Val Leu Leu Thr
Asp Gly Glu Asp Asn 405 410 415 Thr Ile Ser Gly Cys Phe Asn Glu Val
Lys Gln Ser Gly Ala Ile Ile 420 425 430 His Thr Val Ala Leu Gly Pro
Ser Ala Ala Gln Glu Leu Glu Glu Leu 435 440 445 Ser Lys Met Thr Gly
Gly Leu Gln Thr Tyr Ala Ser Asp Gln Val Gln 450 455 460 Asn Asn Gly
Leu Ile Asp Ala Phe Gly Ala Leu Ser Ser Gly Asn Gly 465 470 475 480
Ala Val Ser Gln Arg Ser Ile Gln Leu Glu Ser Lys Gly Leu Thr Leu 485
490 495 Gln Asn Ser Gln Trp Met Asn Gly Thr Val Ile Val Asp Ser Thr
Val 500 505 510 Gly Lys Asp Thr Leu Phe Leu Ile Thr Trp Thr Thr Gln
Pro Pro Gln 515 520 525 Ile Leu Leu Trp Asp Pro Ser Gly Gln Lys Gln
Gly Gly Phe Val Val 530 535 540 Asp Lys Asn Thr Lys Met Ala Tyr Leu
Gln Ile Pro Gly Ile Ala Lys 545 550 555 560 Val Gly Thr Trp Lys Tyr
Ser Leu Gln Ala Ser Ser Gln Thr Leu Thr 565 570 575 Leu Thr Val Thr
Ser Arg Ala Ser Asn Ala Thr Leu Pro Pro Ile Thr 580 585 590 Val Thr
Ser Lys Thr Asn Lys Asp Thr Ser Lys Phe Pro Ser Pro Leu 595 600 605
Val Val Tyr Ala Asn Ile Arg Gln Gly Ala Ser Pro Ile Leu Arg Ala 610
615 620 Ser Val Thr Ala Leu Ile Glu Ser Val Asn Gly Lys Thr Val Thr
Leu 625 630 635 640 Glu Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Thr
Lys Asp Asp Gly 645 650 655 Val Tyr Ser Arg Tyr Phe Thr Thr Tyr Asp
Thr Asn Gly Arg Tyr Ser 660 665 670 Val Lys Val Arg Ala Leu Gly Gly
Val Asn Ala Ala Arg Arg Arg Val 675 680 685 Ile Pro Gln Gln Ser Gly
Ala Leu Tyr Ile Pro Gly Trp Ile Glu Asn 690 695 700 Asp Glu Ile Gln
Trp Asn Pro Pro Arg Pro Glu Ile Asn Lys Asp Asp 705 710 715 720 Val
Gln His Lys Gln Val Cys Phe Ser Arg Thr Ser Ser Gly Gly Ser 725 730
735 Phe Val Ala Ser Asp Val Pro Asn Ala Pro Ile Pro Asp Leu Phe Pro
740 745 750 Pro Gly Gln Ile Thr Asp Leu Lys Ala Glu Ile His Gly Gly
Ser Leu 755 760 765 Ile Asn Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr
Asp His Gly Thr 770 775 780 Ala His Lys Tyr Ile Ile Arg Ile Ser Thr
Ser Ile Leu Asp Leu Arg 785 790 795 800 Asp Lys Phe Asn Glu Ser Leu
Gln Val Asn Thr Thr Ala Leu Ile Pro 805 810 815 Lys Glu Ala Asn Ser
Glu Glu Val Phe Leu Phe Lys Pro Glu Asn Ile 820 825 830 Thr Phe Glu
Asn Gly Thr Asp Leu Phe Ile Ala Ile Gln Ala Val Asp 835 840 845 Lys
Val Asp Leu Lys Ser Glu Ile Ser Asn Ile Ala Arg Val Ser Leu 850 855
860 Phe Ile Pro Pro Gln Thr Pro Pro Glu Thr Pro Ser Pro Asp Glu Thr
865 870 875 880 Ser Ala Pro Cys Pro Asn Ile His Ile Asn Ser Thr Ile
Pro Gly Ile
885 890 895 His Ile Leu Lys Ile Met Trp Lys Trp Ile Gly Glu Leu Gln
Leu Ser 900 905 910 Ile Ala 7 24 DNA Artificial Sequence
Description of Artificial Sequence sense primer for mouse ICACC-1
RNA 7 ccagatccac accaaaacga gaag 24 8 24 DNA Artificial Sequence
Description of Artificial Sequence anti-sense primer for mouse
ICACC-1 RNA 8 cactgtcaaa ggtcaccatc ccga 24 9 20 DNA Artificial
Sequence Description of Artificial Sequence sense primer for human
ICACC-1 RNA 9 gattccagga acagctaagc 20 10 22 DNA Artificial
Sequence Description of Artificial Sequence anti-sense primer for
human ICACC-1 RNA 10 tatttcatag cttgtagcct gg 22 11 21 DNA
Artificial Sequence Description of Artificial Sequence PCR 5'
primer for ICACC-1 11 cccaaaggaa gccaactctg a 21 12 21 DNA
Artificial Sequence Description of Artificial Sequence PCR 3'
primer for ICACC-1 12 gtgaatgcca ggaatggtgc t 21 13 22 PRT
Artificial Sequence Description of Artificial Sequence peptide for
immunization to mICACC-1 13 Cys Leu Val Leu Asp Lys Ser Gly Ser Met
Leu Asn Asp Asp Arg Leu 1 5 10 15 Asn Arg Met Asn Gln Ala 20 14 20
PRT Artificial Sequence Description of Artificial Sequence peptide
for immunization to mICACC-1 14 Gln Ser Glu Leu Lys Gln Leu Asn Ser
Gly Ala Asp Arg Asp Leu Leu 1 5 10 15 Ile Lys His Cys 20 15 25 PRT
Artificial Sequence Description of Artificial Sequence peptide for
immunization to mICACC-1 15 Lys Lys Lys Tyr Pro Thr Asp Gly Ser Glu
Ile Val Leu Leu Thr Asp 1 5 10 15 Gly Glu Asp Asn Thr Ile Ser Ser
Cys 20 25 16 24 PRT Artificial Sequence Description of Artificial
Sequence peptide for immunization to mICACC-1 16 Thr Thr His Pro
Pro Thr Ile Phe Ile Trp Asp Pro Ser Gly Val Glu 1 5 10 15 Gln Asn
Gly Phe Ile Leu Asp Cys 20 17 22 PRT Artificial Sequence
Description of Artificial Sequence peptide for immunization to
mICACC-1 17 Cys Pro Pro Ile Thr Val Thr Pro Val Val Asn Lys Asn Thr
Gly Lys 1 5 10 15 Phe Pro Ser Pro Val Thr 20 18 903 PRT Bos taurus
18 Met Val Pro Arg Leu Thr Val Ile Leu Phe Leu Thr Leu His Leu Leu
1 5 10 15 Pro Gly Met Lys Ser Ser Met Val Asn Leu Ile Asn Asn Gly
Tyr Asp 20 25 30 Gly Ile Val Ile Ala Ile Asn Pro Ser Val Pro Glu
Asp Glu Lys Leu 35 40 45 Ile Gln Asn Ile Lys Glu Met Val Thr Glu
Ala Ser Thr Tyr Leu Phe 50 55 60 His Ala Thr Lys Arg Arg Val Tyr
Phe Arg Asn Val Ser Ile Leu Ile 65 70 75 80 Pro Met Thr Trp Lys Ser
Lys Ser Glu Tyr Leu Met Pro Lys Gln Glu 85 90 95 Ser Tyr Asp Gln
Ala Glu Val Ile Val Ala Asn Pro Tyr Leu Lys His 100 105 110 Gly Asp
Asp Pro Tyr Thr Leu Gln Tyr Gly Arg Cys Gly Glu Lys Gly 115 120 125
Gln Tyr Ile His Phe Thr Pro Asn Phe Leu Leu Thr Asn Asn Leu Pro 130
135 140 Ile Tyr Gly Ser Arg Gly Arg Ala Phe Val His Glu Trp Ala His
Leu 145 150 155 160 Arg Trp Gly Ile Phe Asp Glu Tyr Asn Gly Asp Gln
Pro Phe Tyr Ile 165 170 175 Ser Arg Arg Asn Thr Ile Glu Ala Thr Arg
Cys Ser Thr His Ile Thr 180 185 190 Gly Thr Asn Val Ile Val Lys Cys
Gln Gly Gly Ser Cys Ile Thr Arg 195 200 205 Pro Cys Arg Arg Asp Ser
Gln Thr Gly Leu Tyr Glu Ala Lys Cys Thr 210 215 220 Phe Ile Pro Glu
Lys Ser Gln Thr Ala Arg Glu Ser Ile Met Phe Met 225 230 235 240 Gln
Ser Leu His Ser Val Thr Glu Phe Cys Thr Glu Lys Thr His Asn 245 250
255 Val Glu Ala Pro Asn Leu Gln Asn Lys Met Cys Asn Gly Lys Ser Thr
260 265 270 Trp Asp Val Ile Met Asn Ser Thr Asp Phe Gln Asn Thr Ser
Pro Met 275 280 285 Thr Glu Met Asn Pro Pro Thr Gln Pro Thr Phe Ser
Leu Leu Lys Ser 290 295 300 Lys Gln Arg Val Val Cys Leu Val Leu Asp
Lys Ser Gly Ser Met Ser 305 310 315 320 Ser Glu Asp Arg Leu Phe Arg
Met Asn Gln Ala Ala Glu Leu Phe Leu 325 330 335 Ile Gln Ile Ile Glu
Lys Gly Ser Leu Val Gly Met Val Thr Phe Asp 340 345 350 Ser Val Ala
Glu Ile Arg Asn Asn Leu Thr Lys Ile Thr Asp Asp Asn 355 360 365 Val
Tyr Glu Asn Ile Thr Ala Asn Leu Pro Gln Glu Ala Asn Gly Gly 370 375
380 Thr Ser Ile Cys Arg Gly Leu Lys Ala Gly Phe Gln Ala Ile Ile Gln
385 390 395 400 Ser Gln Gln Ser Thr Ser Gly Ser Glu Ile Ile Leu Leu
Thr Asp Gly 405 410 415 Glu Asp Asn Glu Ile His Ser Cys Ile Glu Glu
Val Lys Gln Ser Gly 420 425 430 Val Ile Ile His Thr Ile Ala Leu Gly
Pro Ser Ala Ala Lys Glu Leu 435 440 445 Glu Thr Leu Ser Asp Met Thr
Gly Gly His Arg Phe Tyr Ala Asn Lys 450 455 460 Asp Ile Asn Gly Leu
Thr Asn Ala Phe Ser Arg Ile Ser Ser Arg Ser 465 470 475 480 Gly Ser
Ile Thr Gln Gln Thr Ile Gln Leu Glu Ser Lys Ala Leu Ala 485 490 495
Ile Thr Glu Lys Lys Trp Val Asn Gly Thr Val Pro Val Asp Ser Thr 500
505 510 Ile Gly Asn Asp Thr Phe Phe Val Val Thr Trp Thr Ile Lys Lys
Pro 515 520 525 Glu Ile Leu Leu Gln Asp Pro Lys Gly Lys Lys Tyr Lys
Thr Ser Asp 530 535 540 Phe Lys Glu Asp Lys Leu Asn Ile His Ser Ala
Arg Leu Arg Ile Pro 545 550 555 560 Gly Ile Ala Glu Thr Gly Thr Trp
Thr Tyr Ser Leu Leu Asn Asn His 565 570 575 Ala Ser Pro Gln Ile Leu
Thr Val Thr Val Thr Thr Arg Ala Arg Ser 580 585 590 Pro Thr Thr Pro
Pro Val Thr Ala Thr Ala His Met Ser Gln Asn Thr 595 600 605 Ala His
Tyr Pro Ser Pro Val Ile Val Tyr Ala Gln Val Ser Gln Gly 610 615 620
Phe Leu Pro Val Leu Gly Ile Asn Val Thr Ala Ile Ile Glu Thr Glu 625
630 635 640 Asp Gly His Gln Val Thr Leu Glu Leu Trp Asp Asn Gly Ala
Gly Ala 645 650 655 Asp Thr Val Lys Asn Asp Gly Ile Tyr Ser Arg Tyr
Phe Thr Asp Tyr 660 665 670 Arg Gly Asn Gly Arg Tyr Ser Leu Lys Val
His Ala Glu Ala Arg Asn 675 680 685 Asn Thr Ala Arg Leu Ser Leu Arg
Gln Pro Gln Asn Lys Ala Leu Tyr 690 695 700 Ile Pro Gly Tyr Ile Glu
Asn Gly Lys Ile Ile Leu Asn Pro Pro Arg 705 710 715 720 Pro Glu Val
Lys Asp Asp Leu Ala Lys Ala Glu Ile Glu Asp Phe Ser 725 730 735 Arg
Leu Thr Ser Gly Gly Ser Phe Thr Val Ser Gly Ala Pro Pro Gly 740 745
750 Asn His Pro Ser Val Leu Pro Pro Asn Lys Ile Ile Asp Leu Glu Ala
755 760 765 Lys Phe Lys Glu Asp His Ile Gln Leu Ser Trp Thr Ala Pro
Ala Asn 770 775 780 Val Leu Asp Lys Gly Lys Ala Asn Ser Tyr Ile Ile
Arg Ile Ser Lys 785 790 795 800 Ser Phe Leu Asp Leu Gln Lys Asp Phe
Asp Asn Ala Thr Leu Val Asn 805 810 815 Thr Ser Ser Leu Lys Pro Lys
Glu Ala Gly Ser Asp Glu Asn Phe Glu 820 825 830 Phe Lys Pro Glu Pro
Phe Arg Ile Glu Asn Gly Thr Asn Phe Tyr Ile 835 840 845 Ala Val Gln
Ala Ile Asn Glu Ala Asn Leu Thr Ser Glu Val Ser Asn 850 855 860 Ile
Ala Gln Ala Ile Lys Phe Ile Pro Met Pro Glu Asp Ser Val Pro 865 870
875 880 Ala Leu Gly Thr Lys Ile Ser Ala Ile Asn Leu Ala Ile Phe Ala
Leu 885 890 895 Ala Met Ile Leu Ser Ile Val 900
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